This work was supported by the National Natural Science Foundation of China (#31771377/ 31571273/31371256), the Foreign Distinguished Scientist Program from the National Department of Education (#MS2014SXSF038), the National Department of Education Central Universities Research Fund (#GK201301001/201701005/GERP-17-45), and XZ is supported by Outstanding Doctoral Thesis Fund (#2019TS082 /2019TS079), Key Program of Shaanxi Provincial Education Department (#20JS138), the Natural Science Basic Research Program Youth Project of Shaanxi Provincial Science and Technology Department (#2020JQ-885).

1. Preparation of grape juice agar plates
<ol>
	<li>Add 5 g of agar and 5 g of sucrose to 150 mL of distilled water. Boil it using a microwave until the agar and sucrose are fully dissolved. Mix 50 mL of 100% grape juice and the solution together.</li>
	<li>Add 1 mL of 100% propionic acid to make the final concentration to 0.5% propionic acid. Pour 25 mL of the prepared solution into each plate. After the agar has solidified, store the media in 4 &#176;C.</li>
</ol>
2. Coating slides
<ol>
	<li>Pretreat slides with detergent. Wash the slides 3 times with tap water and once with distilled water. Then dry the slides in a 45 &#176;C oven for 2 h.</li>
	<li>Immerse the slides in about 450 mL of potassium dichromate sulfuric acid solution (20 g of potassium dichromate, 40 mL of distilled water, and 400 mL of concentrated sulfuric acid) for 24 hours. Wash the slides 3 times with tap water and once with distilled water.
	<ol>
		<li>Dry the slides in a 45 &#176;C oven for two hours. Immerse in 95% ethanol for more than 2 h.</li>
	</ol>
	</li>
	<li>Add 0.5 g of chromium potassium sulfate and 5 g of gelatin to 1 L of distilled water. Dissolve in a 60 &#176;C water bath before use. The chrome alum gelatin can be stored at 4 &#176;C for long periods.</li>
	<li>Drop 10 &#181;L of the chrome alum gelatin on the slide and fully and evenly cover the entire surface of the slide with the solution. Place the slide with the gelatin side up in an oven at 42 &#176;C for 48 h. The prepared slides can be stored at 4 &#176;C for later use. 
	&#8203;NOTE: This is a critical step. The complete coating is necessary. For fruit fly and its embryo, this slide coating approach appears more efficient than poly-lysine coating.</li>
</ol>
3. Drosophila embryo embedding
<ol>
	<li>Drosophila embryo collection 
	NOTE: We collected Drosophila embryos in four periods of 0-2 hours (syncytial blastoderm), 2-3 hours (cellular blastoderm), 3-12 hours (early gastrula) and 12-24 hours (late gastrula)18. The proportion of the main embryo types in each period is shown in Table 1.
	<ol>
		<li>Place 400-500 flies&#160;in a plastic collection bottle and cover the bottle with the grape juice agar plate containing yeast extract. Drosophila embryos are laid on the grape juice agar at room temperature.</li>
		<li>Collect four periods of embryos, gently adding 2 mL of PBS (137 mM NaCl, 2.7 mM KCL, 10 mM Na2HPO4, 2 mM KH2PO4, pH 7.4) to each plate to float the embryos. Transfer about 200 embryos to a 50 mL centrifuge tube and keep it on ice for 2 min to settle down the embryos.</li>
	</ol>
	</li>
	<li>Pre-embedding
	<ol>
		<li>Transfer 200 Drosophila embryos to a 1.5 mL centrifuge tube. Keep the tube on ice for 2 min to settle down the embryos, and then discard the supernatant with pipette carefully. Add 1 mL of 50% bleach in the tube and shake it gently for 2 min. 
		NOTE: The bleach should prepare fresh, and the amount of the bleach could vary depending on the amount of embryos collected.</li>
		<li>Gently collect embryos by pour on filter paper and wash them 3 times with 1 mL of PBS at each time.</li>
		<li>Transfer the embryos with filter paper to about 5 mL of n-heptane.</li>
		<li>Transfer the mixture to a fresh 1.5 mL centrifuge tube. Add 1 mL of methanol and shake it gently. Collect embryos after sedimentation.</li>
		<li>Remove supernatant and wash the embryos in 1 mL of PBS 3-5 times.</li>
		<li>Fix the embryos with 400 &#181;L of Bouin&#39;s solution (71% saturated picric acid, 24% formalin, 5% acetic acid) for 30 min. Wash the fixed embryos 3 times in 1 mL of PBS for 5 min each time.</li>
		<li>To aggregate embryos, transfer the samples into a rubber stopper and remove the PBS.</li>
		<li>Prepare 1.2% agarose-PBS solution and keep solution at 60 &#176;C. Add 200 &#181;L of 1.2% agarose-PBS solution onto embryos to fix them into an agar block.</li>
		<li>Take the block out from stopper and store the block in PBS. 
		NOTE: During pre-embedding approach, pro-warming is critical for successful. We strongly suggest that the centrifuge tubes and micropipette tips, which will be used in this step should be completely pre-warm before operating.</li>
	</ol>
	</li>
	<li>Paraffin embedding
	<ol>
		<li>Immerse the pre-embedded agar block in 35% ethanol, 50% ethanol, 75% ethanol, and 85% ethanol for 15 min each. Then dip 2 times in 95% ethanol and 100% ethanol. 
		NOTES: About 5-10x of block volume of each followed by a graded fixative for 15-30 minutes is necessary depending on number of embryos in the block.</li>
		<li>Immerse the embryos block in 100% ethanol and xylene (1:1 ratio) solution for 15 min.</li>
		<li>Transfer the block to xylene for 1 min to make the embryo tissue transparent.</li>
		<li>Submerge the block in xylene and paraffin (1:1 ratio) for 30 min.</li>
		<li>Submerge the block in paraffin I, paraffin II, and paraffin III for 2 h each time.</li>
		<li>Fill the embedding box with paraffin beforehand, and then gently and horizontally transfer the block into the bottom of embedding box. After the paraffin solidify naturally, the solidified paraffin block can be stored at 4 &#176;C. 
		&#8203;NOTES: The pre-embedded sample can be used in cryosection by following the following steps. After the embryos are pre-embedded with agarose as described above, the samples are simply embedded in OCT Compound. Afterwards, cryosection can be carried out according to routine cryosection protocol. Subsequently, regular staining approaches can be used on the sections.</li>
	</ol>
	</li>
</ol>
4. Hematoxylin-eosin staining
<ol>
	<li>Prepare Drosophila embryo sections. Place prepared sections in an oven at 60 &#176;C for 15 min and then submerge the sections in xylene 2 times for 10 min each. Submerge the sections once in 100% ethanol and xylene (1:1 ratio) for 2 min.</li>
	<li>Hydrate the sections in 100% ethanol I, 100% ethanol II, 95% ethanol, 85% ethanol, 75% ethanol, 50% ethanol, and in distilled water for 3 min.</li>
	<li>Stain the sections with hematoxylin solution for 2 min. Dip the slides in 1% acid alcohol solution (1 mL of hydrochloric acid, 100 mL of 70% ethanol) quickly and wash the slides in distilled water.</li>
	<li>Dip the slides in 50% ethanol, 75% ethanol, 85% ethanol, and 95% ethanol sequentially.</li>
	<li>Stain the sections with eosin solution for 1 min.</li>
	<li>Dehydrate the slides in 95% ethanol I, 95% ethanol II, 95% ethanol III, 100% ethanol I, 100% ethanol II and 100% ethanol III for 1 min each time.</li>
	<li>Clear the slides in 100% xylene I, 100% xylene II, and 100% xylene III for 5 min each time.</li>
	<li>Mount the slides with neutral balsam according to manufacturer&#39;s instructions.</li>
</ol>
5. Periodic acid-silver methenamine staining
<ol>
	<li>Deparaffinize and hydrate paraffin sections of Drosophila embryo to distilled water.</li>
	<li>Oxidize the sections in 1% periodic acid for 15 min at room temperature.</li>
	<li>Rinse the slides in distilled water 3 times for 1 min each.</li>
	<li>Incubate the slides in about 40 mL of freshly filtered silver methenamine working solution (3% methenamine, 5% silver nitrate, 5% sodium borate solution) for 1 h and 20 minutes at 60 &#176;C. Then rinse the slides in distilled water for 1 min.</li>
	<li>Tone the slides in 0.2% gold chloride for 2 min and then rinse the slides in distilled water for 1 min.</li>
	<li>Place the slides in 3% sodium thiosulfate for 3 min and then rinse the slides in distilled water for 1 min.</li>
	<li>Counterstain the slides in hematoxylin for 30 s. Wash the slides in running tap water for 3-5 min.</li>
	<li>Dehydrate the slides in 70% ethanol, 80% ethanol, 95% ethanol I, 95% ethanol II, 95% ethanol III, 100% ethanol I, and 100% ethanol II for 5 min each.</li>
	<li>Clear the slides in 100% xylene I, 100% xylene II and 100% xylene III for 5 min each.</li>
	<li>Mount the slides in neutral balsam according to manufacturer&#39;s instructions.</li>
</ol>
6. Immunohistochemistry
<ol>
	<li>Deparaffinize and rehydrate paraffin sections of Drosophila embryo to PBS following the standard protocol19.</li>
	<li>Treat slides with 0.3% H2O2 in methanol for 10 min at room temperature and avoid light.</li>
	<li>Pre-heat the appropriate antigen retrieval buffer to approximate 100 &#176;C in a container with the slides in the vegetable steamer. The container should also have a lid. Put the slides in the container. Close the lid of the steamer.
	<ol>
		<li>Keep the container in the steamer for 20 minutes from this point. Note that the lid of the slide container should not be entirely tightened so water vapor can enter during the steaming process.</li>
	</ol>
	</li>
	<li>Allow the slides return to room temperature before gently rinsing it in PBS-T (PBS with Tween-20, pH 7.2) 3 times for 5 min each, and then rinse the slides in PBS 3 times for 1 min each.</li>
	<li>Block the slides with 1% bovine serum albumin (BSA) in PBS for 60 min at room temperature.</li>
	<li>Incubate the slides with primary antibody (anti-FKBP12 at 1:1000) overnight at 4 &#176;C or at room temperature for 4 h.</li>
	<li>Wash the slides in PBS-T 4 times for 5 min each.</li>
	<li>Apply the slides with HRP labeled polymer conjugated with secondary antibodies (anti-Rabbit, 1:5,000) for 90 min at room temperature. Do this in the dark to avoid photo bleaching.</li>
	<li>Wash the slides in PBS-T 3 times for 5 min each.</li>
	<li>Incubate the slides with 5% 3,3&#39;-diaminobenzidine-tetrahydrochloride (DAB) for 2-10 min until the color of reaction products is appropriate under microscope.</li>
	<li>Counterstain the slides in hematoxylin for 30 s and wash the slides in distilled water 2 times for 5 min each.</li>
	<li>Dehydrate the slides in 50% ethanol, 70% ethanol, 90% ethanol, 95% ethanol, and 100% ethanol for 1 min each.</li>
	<li>Clear the slides in 100% xylene I, 100% xylene II and 100% xylene III for 5 min each.</li>
	<li>Mount the slides in neutral balsam according to manufacturer&#39;s instructions.</li>
</ol>
7. In-situ hybridization
NOTE: Drosophila embryo sections were prepared with 0.01% diethyl pyrocarbonate (DEPC) treated water. All accessories used for in situ hybridization apply DEPC overnight treatment in advance.
<ol>
	<li>Riboprobe preparation
	<ol>
		<li>Linearize template DNA by cutting at a restriction enzyme site downstream from the cloned insert using a restriction enzyme that creates 5&#39; overhangs. After linearization, purify the template DNA via phenol/chloroform extraction, and subsequent ethanol precipitation. Use standard protocols.</li>
		<li>Add 1 &#181;g of purified template DNA to a RNase-free centrifuge tube. Place the centrifuge tube on ice. Add enough DEPC-treated water to the total volume 13 &#181;L.
		<ol>
			<li>Then add the following reagents in order: 2 &#181;L of 10x NTP labeling mixture, 2 &#181;L of 10x transcription buffer, 1 &#181;L of protector RNase inhibitor, 2 &#181;L of RNA Polymerase SP6 or RNA Polymerase T7.</li>
		</ol>
		</li>
		<li>Mix the reaction by pipetting and centrifuge shortly. Incubate for 2 h at 37 &#176;C. 
		NOTE: Longer incubations do not increase the yield of labeled RNA. A short spin (~800 rpm) applied on the reaction tube is critical necessary before continuing to the next step. This spin will allow all contents, which could generate from the incubation, to come to the bottom of the tube.</li>
		<li>Add 2 &#181;L of RNase-free DNase I to remove template DNA and incubate for 15 min at 37 &#176;C.</li>
		<li>Add 2 &#181;L of 200 mM EDTA (pH 8.0) to stop the reaction. 
		NOTE: DIG-labeled RNA probes can be stored at -15 to -25 &#176;C in ethanol for one year.</li>
	</ol>
	</li>
	<li>Pre-hybridization of Drosophila embryo section
	<ol>
		<li>Place sections of Drosophila embryo in a 42 &#176;C oven for 48 h.</li>
		<li>Deparaffinize the slides in 100% xylene for 10 min twice.</li>
		<li>Hydrate the slides by sequentially submerging in 100% ethanol, 95% ethanol, 90% ethanol, 70% ethanol, and 50% ethanol for 5 min.</li>
		<li>Incubate the slides in PBS for 5 min 3 times to remove the fixative solution/Bouin&#39;s solution from the tissue.</li>
		<li>Wash the slides with 100 mmol/L glycine/PBS solution 2 times for 5 min each.</li>
		<li>Wash the slides with PBS 2 times for 5 min each.</li>
		<li>Incubate the slides with 0.3% Triton X-100 in PBS for 15 min to permeabilize the membranes.</li>
		<li>Wash the slides with PBS 3 times for 5 min each.</li>
		<li>Incubate the slides in 15 &#181;g/mL RNase-free proteinase K for 30 minutes at 37 &#176;C.</li>
		<li>Incubate the slides with 4% paraformaldehyde in PBS for 3 min to stop the digestion.</li>
		<li>Wash the slides with PBS 2 times for 5 min each.</li>
		<li>Incubate the slides in 100 mM triethanolamine buffer (pH 8.0) with 0.25% (v/v) acetic anhydride 2 times for 5 min each.</li>
		<li>Add DEPC water into the slide moisture chamber. Add 20 &#181;L of pre-hybridization solution (containing 100 &#181;g/mL of salmon sperm DNA) on the slides. Put the slides into the slide moisture chamber. Incubate the slides at 42 &#176;C for 4 h. 
		NOTE: It is necessary to ensure the tightness of the lid of the wet box and that the wet box is always kept in a horizontal to prevent volatilization and deviation from the tissue of the pre-hybridization solution and the followed hybridization solution.</li>
	</ol>
	</li>
	<li>Hybridization of Drosophila embryo section
	<ol>
		<li>Remove the pre-hybridization solution and wash the slides with 0.2x SSC (150 mM sodium chloride, 15 mM sodium citrate, pH 7.0) 3 times for 5 min each. Wipe off excess 0.2x SSC around the tissue samples.</li>
		<li>Apply 30 &#181;L of probe hybridization solution (containing 2-6 ng of digoxin-labeled RNA probe by diluting in pre-hybridization solution) with a pipette. Incubate the slides in the slide moisture chamber at 42 &#176;C for approximately 20 h. 
		NOTE: Be sure on the cap-tightness as the suggestion of the last note. Otherwise, the dry-up caused from the leakage of either pre-hybridization or pre-hybridization will generate dirty background including pseudo-positive in slides.</li>
	</ol>
	</li>
	<li>Hybridization
	<ol>
		<li>Remove the hybridization solution and wash the slides with 4x SSC solution 4 times for 15 min each at 37 &#176;C.</li>
		<li>Wash the slides in 2x SSC solution with 20 &#181;g/mL of RNase A for 30 min at 37 &#176;C.</li>
		<li>Wash the slides in 1x SSC solution for 15 min at 37 &#176;C.</li>
		<li>Wash the slides in 0.5x SSC solution for 15 min at 37 &#176;C.</li>
		<li>Wash the slides in 0.1x SSC solution for 15 min at 37 &#176;C.</li>
		<li>Wash the slides with PBS 3 times for 5 min each.</li>
		<li>Wash the slides in washing buffer (100 mM maleic acid, 150 mM NaCl, 0.3% (v/v) Tween 20, pH 7.5) for 1 min.</li>
		<li>Incubate the slides in 100 mL of freshly prepared blocking solution (2% sheep serum in maleic acid buffer without Tween 20) for 30 minutes.</li>
		<li>Incubate the slides in 20 mL of antibody solution for 45 minutes.
		<ol>
			<li>Centrifuge the antibody for 5 min at 10,000 rpm in the original vial prior to each use, and pipet the necessary amount carefully from the surface. Dilute anti-digoxigenin-AP 1:5000 (150 mU/mL) in blocking solution.</li>
		</ol>
		</li>
		<li>Wash the slides in 100 mL of washing buffer 2 times for 15 minutes each to remove unbound antibody conjugate.</li>
		<li>Equilibrate the slides in 20 mL of detection buffer (100 mM Tris-HCl, 100 mM NaCl, pH 9.5) for 2-5 min.</li>
		<li>Incubate the slides with 10 mL of freshly prepared color substrate solution in the slide moisture chamber. The reaction begins within a few minutes and is complete after 16 h. Do not shake or mix while color is developing.</li>
		<li>Wash the slides with 50 mL of distilled water for 5 min to stop the reaction.</li>
		<li>Dry the sample overnight in the dark.</li>
		<li>Dehydrate the slides in 70% ethanol, 80% ethanol, 90% ethanol, 95% ethanol, 100% ethanol I and 100% ethanol II for 3 min each.</li>
		<li>Clear the slides in 100% xylene I, 100% xylene II and 100% xylene III for 20 min each.</li>
		<li>Mount the slides with neutral balsam.</li>
		<li>Document images and analyze with the inverted microscopy and manufacturer software. Perform distribution analysis using ImageJ according to density of positive signal either along longitudinal axis or within differential zone of embryos. 
		NOTE: During the incubation with the freshly prepared color substrate solution in the step 7.4.12, the reaction color can be seen even with a bare eye. This observation on how color developing can be inspected under stereoscope. Once reaction color is reasonable and the step 7.4.13 can be carried out to stop the reaction.</li>
	</ol>
	</li>
</ol>

<ol><li>Weisleder, N., Ma, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Altered+Ca2%2B+sparks+in+aging+skeletal+and+cardiac+muscle%5BTitle%5D)+AND+%22Ageing+Research+Reviews%22%5BJournal%5D)">Altered Ca2+ sparks in aging skeletal and cardiac muscle.</a> Ageing Research Reviews. 7 (3), 177-188 (2008).</li>
<li>Cheng, H., Lederer, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Calcium+Sparks%5BTitle%5D)+AND+%22Physiological+Reviews%22%5BJournal%5D)">Calcium Sparks.</a> Physiological Reviews. 88 (4), 1491-1545 (2008).</li>
<li>Fan, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ryanodine+Receptors%3A+Functional+Structure+and+Their+Regulatory+Factors%5BTitle%5D)+AND+%22Chinese+Journal+of+Cell+Biology%22%5BJournal%5D)">Ryanodine Receptors: Functional Structure and Their Regulatory Factors.</a> Chinese Journal of Cell Biology. 37 (1), 6-15 (2015).</li>
<li>Xu, X., Balk, S. P., Isaacs, W., Ma, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Calcium+signaling%3A+an+underlying+link+between+cardiac+disease+and+carcinogenesis%5BTitle%5D)+AND+%22Cell+%26+Bioscience%22%5BJournal%5D)">Calcium signaling: an underlying link between cardiac disease and carcinogenesis.</a> Cell & Bioscience. 8 (39), 1-2 (2018).</li>
<li>Xu, X., Bhat, M. B., Nishi, M., Takeshima, H., Ma, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Molecular+cloning+of+cDNA+encoding+a+Drosophila+ryanodine+receptor+and+functional+studies+of+the+carboxyl-terminal+calcium+Release+Channel%5BTitle%5D)+AND+%22Biophysical+Journal%22%5BJournal%5D)">Molecular cloning of cDNA encoding a Drosophila ryanodine receptor and functional studies of the carboxyl-terminal calcium Release Channel.</a> Biophysical Journal. 78 (3), 1270-1281 (2000).</li>
<li>George, G. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comparative+analysis+of+FKBP+family+protein%3A+evaluation%2C+structure%2C+and+function+in+mammals+and+Drosophila+melanogaster%5BTitle%5D)+AND+%22BMC+Developmental+Biology%22%5BJournal%5D)">Comparative analysis of FKBP family protein: evaluation, structure, and function in mammals and Drosophila melanogaster.</a> BMC Developmental Biology. 18 (1), 1-12 (2018).</li>
<li>Zhou, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Syncytium+calcium+signaling+and+macrophage+function+in+the+heart%5BTitle%5D)+AND+%22Cell+%26+Bioscience%22%5BJournal%5D)">Syncytium calcium signaling and macrophage function in the heart.</a> Cell & Bioscience. 8 (1), 1-9 (2018).</li>
<li>Wang, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Calcium+and+CaSR%2FIP3R+in+prostate+cancer+development%5BTitle%5D)+AND+%22Cell+%26+Bioscience%22%5BJournal%5D)">Calcium and CaSR/IP3R in prostate cancer development.</a> Cell & Bioscience. 8 (1), 1-7 (2018).</li>
<li>Xu, M., Seas, A., Kiyani, M., Ji, K. S., Bell, H. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+temporal+examination+of+calcium+signaling+in+cancer-+from+tumorigenesis%2C+to+immune+evasion%2C+and+metastasis%5BTitle%5D)+AND+%22Cell+%26+Bioscience%22%5BJournal%5D)">A temporal examination of calcium signaling in cancer- from tumorigenesis, to immune evasion, and metastasis.</a> Cell & Bioscience. 8 (1), 1-9 (2018).</li>
<li>Shou, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cardiac+defects+and+altered+ryanodine+receptor+function+in+mice+lacking+FKBP12%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Cardiac defects and altered ryanodine receptor function in mice lacking FKBP12.</a> Nature. 391 (6666), 489-492 (1998).</li>
<li>Xin, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Oestrogen+protects+FKBP12.6+null+mice+from+cardiac+hypertrophy%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Oestrogen protects FKBP12.6 null mice from cardiac hypertrophy.</a> Nature. 416 (6878), 334-337 (2002).</li>
<li>Zalk, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Structure+of+a+mammalian+ryanodine+receptor%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Structure of a mammalian ryanodine receptor.</a> Nature. 517 (7532), 44-49 (2015).</li>
<li>Ridgway, E. B., Gilkey, J. C., Jaffe, L. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Free+calcium+increases+explosively+in+activating+medaka+eggs%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">Free calcium increases explosively in activating medaka eggs.</a> Proceedings of the National Academy of Sciences of the United States of America. 74 (2), 623-627 (1977).</li>
<li>Gilkey, J. C., Jaffe, L. F., Ridgway, E. B., Reynolds, G. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+free+calcium+wave+traverses+the+activating+egg+of+the+medaka%2C+Oryzias+latipes%5BTitle%5D)+AND+%22Journal+of+Cell+Biology%22%5BJournal%5D)">A free calcium wave traverses the activating egg of the medaka, Oryzias latipes.</a> Journal of Cell Biology. 76 (2), 448-466 (1978).</li>
<li>Kreko-Pierce, T., Azpurua, J., Mahoney, R. E., Eaton, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Extension+of+health+span+and+life+span+in+Drosophila+by+S107+requires+the+calstabin+homologue+FK506-BP2%5BTitle%5D)+AND+%22Journal+of+Biological+Chemistry%22%5BJournal%5D)">Extension of health span and life span in Drosophila by S107 requires the calstabin homologue FK506-BP2.</a> Journal of Biological Chemistry. 291 (50), 26045-26055 (2016).</li>
<li>Sullivan, K. M., Scott, K., Zuker, C. S., Rubin, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+ryanodine+receptor+is+essential+for+larval+development+in+Drosophila+melanogaster%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">The ryanodine receptor is essential for larval development in Drosophila melanogaster.</a> Proceedings of the National Academy of Sciences of the United States of America. 97 (11), 5942-5947 (2000).</li>
<li>Feng, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dynamics+expression+of+DmFKBP12%2FCalstabin+during+embryonic+early+development+of+Drosophila+melanogaster%5BTitle%5D)+AND+%22Cell+%26+Bioscience%22%5BJournal%5D)">Dynamics expression of DmFKBP12/Calstabin during embryonic early development of Drosophila melanogaster.</a> Cell & Bioscience. 9 (1), 1-16 (2019).</li>
<li>Campos-Ortega, J. A., Hartenstein, V. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aCampos-Ortega%2C+JA+%22The+embryonic+development+of+Drosophila+melanogaster%22">The embryonic development of Drosophila melanogaster</a>. , Springer. 633-645 (1958).</li>
<li>Xu, X., Dong, C., Vogel, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hemicentins+Assemble+on+Diverse+Epithelia+in+the+Mouse%5BTitle%5D)+AND+%22Journal+of+Histochemistry+and+Cytochemistry%22%5BJournal%5D)">Hemicentins Assemble on Diverse Epithelia in the Mouse.</a> Journal of Histochemistry and Cytochemistry. 55 (2), 119-126 (2007).</li>
<li>Wehrens, X. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((FKBP12.6+deficiency+and+defective+calcium+release+channel+%28ryanodine+receptor%29+function+linked+to+exercise-induced+sudden+cardiac+death%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">FKBP12.6 deficiency and defective calcium release channel (ryanodine receptor) function linked to exercise-induced sudden cardiac death.</a> Cell. 113 (7), 829-840 (2003).</li>
<li>Wehrens, X. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Protection+from+cardiac+arrhythmia+through+ryanodine+receptor-stabilizing+protein+calstabin2%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Protection from cardiac arrhythmia through ryanodine receptor-stabilizing protein calstabin2.</a> Science. 304 (5668), 292-296 (2004).</li>
<li>Bellinger, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Remodeling+of+ryanodine+receptor+complex+causes+%26%2334%3Bleaky%26%2334%3B+channels%3A+a+molecular+mechanism+for+decreased+exercise+capacity%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">Remodeling of ryanodine receptor complex causes &#34;leaky&#34; channels: a molecular mechanism for decreased exercise capacity.</a> Proceedings of the National Academy of Sciences of the United States of America. 105 (6), 2198-2202 (2008).</li>
<li>Maruyama, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((FKBP12+is+a+critical+regulator+of+the+heart+rhythm+and+the+cardiac+voltage-gated+sodium+current+in+mice%5BTitle%5D)+AND+%22Circulation+Research%22%5BJournal%5D)">FKBP12 is a critical regulator of the heart rhythm and the cardiac voltage-gated sodium current in mice.</a> Circulation Research. 108 (9), 1042-1052 (2011).</li>
<li>Xu, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((FKBP12+is+the+only+FK506+binding+protein+mediating+T-cell+inhibition+by+the+immunosuppressant+FK506%5BTitle%5D)+AND+%22Transplantation%22%5BJournal%5D)">FKBP12 is the only FK506 binding protein mediating T-cell inhibition by the immunosuppressant FK506.</a> Transplantation. 73 (11), 1835-1838 (2002).</li>
<li>Zalk, R., Marks, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ca2%2B+release+channels+join+the+%27resolution+revolution%27%5BTitle%5D)+AND+%22Trends+in+Biochemical+Sciences%22%5BJournal%5D)">Ca2+ release channels join the 'resolution revolution'.</a> Trends in Biochemical Sciences. 42 (7), 543-555 (2017).</li>
</ol>

The authors have nothing to disclose.

RyRs and IP3Rs mediated calcium signaling is a fundamental pathway in many physiological and pathological process of both vertebrate and invertebrate animals1,2,3,4. In humans, point mutations, such as CPVT-associated R4496C mutation, in the RyR2 gene lead to calcium leakage from the sarcoplasmic reticulum of cardiomyocyte, resulting in cardiac dysfunction. These mutations present as arrhythmia...

Calcium induced calcium release signaling (CICR) plays critical role in many biological processes, such as skeletal/smooth muscle and cardiac vascular function, cell proliferation and apoptosis, development, aging, neuronal synaptic plasticity, and regeneration1,2,3,4,5,6. Ryanodine receptors (RyRs) and inositol 1,4,5-trisphosphate receptors (IP3Rs) are major players in the calcium signaling pathway controlled by their regulators protein kinase A (PKA), Ca2+/calmodulin-dependent protein kinase II (CaMKII), FK506 binding proteins (FKBPs), calsequestrin (CSQ), triadin, and junctin1,2,3,4,5,6. Abnormal human expression and mutations in these proteins can lead to pathological physiology such as arrhythmias7 and oncogenic proliferation8,9.
FKBPs regulate the calcium release from the endoplasmic reticular (ER) by RyRs. This process is essential for the mechanism of contraction,&#160;and thus responsible for all mechanical movement generated by myosin-actin contraction through calcium-induced calcium release along with embryonic RyRs1,2. In mouse models, the lack of RyR2 and its regulator FKBP12/Calstabin is invariably lethal, either during embryonic development or in the early postnatal period10,11,12. FKBP12/Calstabin knockout mice exhibit critical cardiac defects with irregular excitation- contraction coupling (EC) and cerebral edema during embryonic development. This indicates that FKBP12/Calstabin plays an essential role in regulating RyR2 channel expression, which is important both to cardiac and cerebral development10.
RyR conducted calcium sparks were initially discovered in the zygote formation phase of fertilized Medaka eggs13,14. However, few investigations have been performed on the function of calcium signaling in early embryonic development. In Drosophila melanogaster, results obtained from DmFKBP12 S107 mutants provide strong evidence supporting the importance of this gene for larval development and a healthy life span, which is attributed to its function against oxidative stress15,16. Recently, we identified the dynamic localization of FKBP12/Calstabin protein and messenger RNA during early Drosophila melanogaster development17. Using the approaches described in this methodology, we were able to track the expression of FKBP12/Calstabin in D. melanogaster during the syncytial blastoderm (0-2 h), cellular blastoderm (2-3 h), early gastrula (3-12 h), and late gastrula (12-24 h). In this paper, we present the detailed protocols of every approach in the previous study, including pre-embryo embedding for classic paraffin sectioning, pre-coating slide treatment for embryonic sections, histo-chemistry staining and immunostaining, and mRNA in-situ hybridization for identification of gene expression.

<table><tbody><tr><td>-20&amp;deg;C Refrigerator</td><td>Meiling Biology &amp;amp;Medical</td><td>DW-YL270</td><td>Used for regent storage</td></tr><tr><td>-80&amp;deg;C Ultra low temperature refrigerator</td><td>Thermo</td><td>Forma 90 Series</td><td>Used for regent storage</td></tr><tr><td>Agar</td><td>Sigma-Aldrich</td><td>WXBB6360V</td><td>Preparation of grape juice agar plates</td></tr><tr><td>Anti-Digoxigenin-AP, Fab fragments</td><td>Roche</td><td>11093274910</td><td>For the detection of digoxigenin-labeled compound</td></tr><tr><td>Biochemical incubator</td><td>Shanghai Bluepard Instruments</td><td>LRH-250</td><td>&lt;em&gt;In-situ &lt;/em&gt;Hybridization</td></tr><tr><td>Bouin&#039;s solution</td><td>Sinopharm Chemical Reagent at Beijing</td><td>69945460</td><td>Drosophila Embryo Embedding</td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>540BH07808</td><td>&lt;em&gt;In-situ&lt;/em&gt; Hybridization</td></tr><tr><td>Centrifuge tube</td><td>Denville</td><td>C-2170</td><td>Drosophila Embryo Collection</td></tr><tr><td>Chrome Alum</td><td>Sinopharm Chemical Reagent at Beijing</td><td>10001018</td><td>Coating Slides</td></tr><tr><td>Constant temperature water bath</td><td>Jintan Henfeng Instruments</td><td>KW-1000DC</td><td>Hematoxylin-Eosin Staining, Immunohistochemistry, In-situ Hybridization and Periodic Acid-Silver Methenamine Staining</td></tr><tr><td>Dako REAL EnVision Detection System</td><td>Dako</td><td>K5007</td><td>In immunohistochemical reaction or in situ hybridization reaction, it binds to the primary antigen antibody, and the target is labeled by staining.</td></tr><tr><td>DEPC</td><td>Sigma-Aldrich</td><td>D5758</td><td>In-situ Hybridization</td></tr><tr><td>DIG RNA Labeling Kit</td><td>Roche</td><td>11093274910</td><td>RNA labeling with diagoxigenin-UTP by in vitro transcription with SP6 and T7 RNA polymerase</td></tr><tr><td>&lt;em&gt;Drosophila melanogaster&lt;/em&gt;</td><td>Bloomington Stock Center</td><td>BDSC_16799, BDSC_19894, BDSC_11664</td><td>The stocks of &lt;em&gt;Drosophila melanogaster &lt;/em&gt;mutant</td></tr><tr><td>Electric blast drying oven</td><td>Tianjin Taiste Instruments</td><td>101-0AB</td><td>For coating slides and paraffin embedding</td></tr><tr><td>Eosin</td><td>Sigma-Aldrich</td><td>230251</td><td>Hematoxylin-Eosin Staining</td></tr><tr><td>Ethanol</td><td>Sinopharm Chemical Reagent at Beijing</td><td>100092680</td><td>Paraffin Embedding, Hematoxylin-Eosin Staining, Immunohistochemistry, In-situ Hybridization and Periodic Acid-Silver Methenamine Staining</td></tr><tr><td>Gelatin</td><td>Sinopharm Chemical Reagent at Beijing</td><td>10010328</td><td>Coating Slides</td></tr><tr><td>Gold chloride</td><td>Sigma-Aldrich</td><td>379948</td><td>Periodic Acid-Silver Methenamine Staining</td></tr><tr><td>Hematoxylin</td><td>Sigma-Aldrich</td><td>H3136</td><td>Hematoxylin-Eosin Staining</td></tr><tr><td>High Pure PCR Product Purification Kit</td><td>Roche</td><td>11732668001</td><td>For purification of PCR products</td></tr><tr><td>Intelligent constant temperature and humidity box</td><td>Ningbo Jiangnan Instruments</td><td>HWS</td><td>For fly maintenance</td></tr><tr><td>LE Agarose</td><td>HyAgarose</td><td>14190108029</td><td>Pre-embedding</td></tr><tr><td>Methanol</td><td>Sinopharm Chemical Reagent at Beijing</td><td>10014108</td><td>Drosophila Embryo Collection</td></tr><tr><td>Microscope</td><td>ZEISS</td><td>Observer.A1</td><td>Hematoxylin-Eosin Staining, Immunohistochemistry, In-situ Hybridization and Periodic Acid-Silver Methenamine Staining</td></tr><tr><td>Microscope Slides</td><td>MeVid Labware Manufacturing</td><td>P105-2001</td><td>Coating Slides</td></tr><tr><td>Neutral Gum</td><td>Sinopharm Chemical Reagent at Beijing</td><td>10004160</td><td>Hematoxylin-Eosin Staining</td></tr><tr><td>N-heptane</td><td>Sinopharm Chemical Reagent at Beijing</td><td>40026768</td><td>Drosophila Embryo Collection</td></tr><tr><td>Paraffin slicer</td><td>Huahai science instrument</td><td>HH-2508III</td><td>&lt;em&gt;In-situ&lt;/em&gt; Hybridization</td></tr><tr><td>Paraffin</td><td>Sinopharm Chemical Reagent at Beijing</td><td>69019461</td><td>Paraffin Embedding</td></tr><tr><td>pH/mV Meter</td><td>Sartorius</td><td>PB-10</td><td>For determing the pH value of a solution</td></tr><tr><td>Silver nitrate</td><td>Sinopharm Chemical Reagent at Beijing</td><td>10018461</td><td>Periodic Acid-Silver Methenamine Staining</td></tr><tr><td>Ultrapure water meter</td><td>Thermo</td><td>AFXI-0501-P</td><td>&lt;em&gt;In-situ&lt;/em&gt; Hybridization</td></tr><tr><td>Xylene</td><td>Sinopharm Chemical Reagent at Beijing</td><td>10023418</td><td>Paraffin Embedding</td></tr></tbody></table>

a protocol for immunohistochemistry and rna in-situ distribution within early drosophila embryo

Calcium induced calcium release signaling (CICR) plays a critical role in many biological processes. Every cellular activity from cell proliferation and apoptosis, development and ageing, to neuronal synaptic plasticity and regeneration have been associated with Ryanodine receptors (RyRs). Despite the importance of calcium signaling, the exact mechanism of its function in early development is unclear. As an organism with a short gestational period, the embryos of Drosophila melanogaster are prime study subjects for investigating the distribution and localization of CICR associated proteins and their regulators during development. However, because of their lipid-rich embryos and chitin-rich chorion, their utility is limited by the difficulty of mounting embryos on glass surfaces. In this work, we introduce a practical protocol that significantly enhances the attachment of Drosophila embryo onto slides and detail methods for successful histochemistry, immunohistochemistry, and in-situ hybridization. The chrome alum gelatin slide-coating method and embryo pre-embedding method dramatically increases the yield in studying Drosophila embryo protein and RNA expression. To demonstrate this approach, we studied DmFKBP12/Calstabin, a well-known regulator of RyR during early embryonic development of Drosophila melanogaster. We identified DmFKBP12 in as early as the syncytial blastoderm stage and report the dynamic expression pattern of DmFKBP12 during development: initially as an evenly distributed protein in the syncytial blastoderm, then preliminarily localizing to the basement layer of the cortex during cellular blastoderm, before distributing in the primitive neuronal and digestion architecture during the three-gem layer phase in early gastrulation. This distribution may explain the critical role RyR plays in the vital organ systems that originate in from these layers: the suboesophageal and supraesophageal ganglion, ventral nervous system, and musculoskeletal system.

The figures describe protocols used to overcome the challenge of attaching high lipid and chitin-containing chorion Drosophila embryos (Table 1) to the glass slide surface for examination and experimentation. Utilizing the chrome alum gelatin slide-coating method shown in Figure 1, we enhanced the attachment of Drosophila embryos onto the surface of slides while the embryo pre-embedding method shown in Figure 2 allows for effic...

Watch this Scientific Journal Video about A Protocol for Immunohistochemistry and RNA In-situ Distribution within Early Drosophila Embryo at JoVE.com

A Protocol for Immunohistochemistry and RNA In-situ Distribution within Early Drosophila Embryo

Here, we describe a protocol for detection and localization of Drosophila embryo protein and RNA from collection to pre-embedding and embedding, immunostaining, and mRNA in situ hybridization.

A Protocol for Immunohistochemistry and RNA In-situ Distribution within Early Drosophila Embryo

Calcium induced calcium release signaling (CICR) plays a critical role in many biological processes. Every cellular activity from cell proliferation and ...

a-protocol-for-immunohistochemistry-rna-situ-distribution-within

Research

JoVE Journal

Developmental Biology

3.6K Views.  Shaanxi Normal University College of Life Sciences. Als een organisme met een korte draagtijd zijn de embryo's van Drosophila melanogaster de belangrijkste proefpersonen voor het onderzoeken van de distributie en lokalisatie van eiwitten en hun regulatoren tijdens de ontwikkeling. Vanwege hun lipiderijke embryo's en chitinerijke chorion wordt hun nut echter beperkt door de moeilijkheid om embryo's op glazen oppervlakken te monteren. In dit werk introduceren we een praktische methode die de hechting van drosophila-embryo op dia's aanzienlijk ve...

Video: A Protocol for Immunohistochemistry and RNA In-situ Distribution within Early Drosophila Embryo

High Resolution Fluorescent In Situ Hybridization in Drosophila Embryos and Tissues Using Tyramide Signal Amplification

In our efforts to determine the patterns of expression and subcellular localization of Drosophila RNAs on a genome-wide basis, and in a variety of tissues, we have developed numerous modifications and improvements to our original fluorescent in situ hybridization (FISH) protocol. To facilitate throughput and cost effectiveness, all steps, from probe generation to signal detection, are performed using exon 96-well microtiter plates. Digoxygenin (DIG)-labelled antisense RNA probes are produced using either cDNA clones or genomic DNA as templates. After tissue fixation and permeabilization, probes are hybridized to transcripts of interest and then detected using a succession of anti-DIG antibody conjugated to biotin, streptavidin conjugated to horseradish peroxidase (HRP) and fluorescently conjugated tyramide, which in the presence of HRP, produces a highly reactive intermediate that binds to electron dense regions of immediately adjacent proteins. These amplification and localization steps produce a robust and highly localized signal that facilitates both cellular and subcellular transcript localization. The protocols provided have been optimized to produce highly specific signals in a variety of tissues and developmental stages. References are also provided for additional variations that allow the simultaneous detection of multiple transcripts, or transcripts and proteins, at the same time.

High Resolution Fluorescent In Situ Hybridization in Drosophila Embryos and Tissues Using Tyramide Signal Amplification

The described RNA in situ hybridization protocol allows the detection of RNA in whole Drosophila embryos or dissected tissues. Using 96-well microtiter plates and tyramide signal amplification, transcripts can be detected at high resolution, sensitivity, and throughput, and at a relatively low cost.

In our efforts to determine the patterns of expression and subcellular localization of Drosophila RNAs on a genome-wide basis, and in a variety of ...

Genetics

Multi-target Chromogenic Whole-mount In Situ Hybridization for Comparing Gene Expression Domains in Drosophila Embryos

To analyze gene regulatory networks active during embryonic development and organogenesis it is essential to precisely define how the different genes are expressed in spatial relation to each other in situ. Multi-target chromogenic whole-mount in situ hybridization (MC-WISH) greatly facilitates the instant comparison of gene expression patterns, as it allows distinctive visualization of different mRNA species in contrasting colors in the same sample specimen. This provides the possibility to relate gene expression domains topographically to each other with high accuracy and to define unique and overlapping expression sites. In the presented protocol, we describe a MC-WISH procedure for comparing mRNA expression patterns of different genes in Drosophila embryos. Up to three RNA probes, each specific for another gene and labeled by a different hapten, are simultaneously hybridized to the embryo samples and subsequently detected by alkaline phosphatase-based colorimetric immunohistochemistry. The described procedure is detailed here for Drosophila, but works equally well with zebrafish embryos.

Multi-target Chromogenic Whole-mount In Situ Hybridization for Comparing Gene Expression Domains in Drosophila Embryos

We describe a multi-target chromogenic whole-mount in situ hybridization (MC-WISH) procedure in intact Drosophila embryos allowing simultaneous and specific detection of three different mRNA distribution patterns by contrasting color precipitates.

To analyze gene regulatory networks active during embryonic development and organogenesis it is essential to precisely define how the different genes are ...

Drosophila Larval NMJ Immunohistochemistry

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use of the Drosophila motor system is due to its high accessibility. It can be analyzed with single-cell resolution. There are 30 muscles per hemisegment whose arrangement within the peripheral body wall are known. A total of 31 motor neurons attach to these muscles in a pattern that has high fidelity. Using molecular biology and genetics, one can create transgenic animals or mutants. Then, one can study the developmental consequences on the morphology and function of the NMJ. Immunohistochemistry can be used to clearly image the components of the NMJ. In this article, we demonstrate how to use antibody staining to visualize the Drosophila larval NMJ.

Drosophila Larval NMJ Immunohistochemistry

This protocol demonstrates how to perform immunohistochemistry on dissected Drosophila larva.

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use ...

Biology

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the proteins involved in these pathways are evolutionarily conserved between mammals and other eukaryotes, such as the fruit fly Drosophila melanogaster, suggesting that similar organizing principles exist during the development of these organisms. Importantly, Drosophila has been used extensively to identify cellular and molecular mechanisms regulating processes that are required in mammals including neurogenesis, differentiation, axonal guidance, and synaptogenesis. Flies have also been used successfully to model a variety of human neurodevelopmental diseases. Here we describe a protocol for the step-by-step microdissection, fixation, and immunofluorescent localization of proteins within the adult Drosophila brain. This protocol focuses on two example neuronal populations, mushroom body neurons and retinal photoreceptors, and includes optional steps to trace individual mushroom body neurons using Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. Example data from both wild-type and mutant brains are shown along with a brief description of a scoring criteria for axonal guidance defects. While this protocol highlights two well-established antibodies for investigating the morphology of mushroom body and photoreceptor neurons, other Drosophila brain regions and the localization of proteins within other brain regions can also be investigated using this protocol.

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

This protocol describes the dissection and immunostaining of adult Drosophila melanogaster brain tissues. Specifically, this protocol highlights the use of Drosophila mushroom body and photoreceptor neurons as example neuronal subsets that can be accurately used to uncover general principles underlying many aspects of neuronal development.

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the ...

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. The protocol is a modified version of the standard in situ hybridization using alkaline phosphatase and substrates such as NBT/BCIP and Fast Red 1,2. This protocol utilizes standard digoxygenin and fluorescein labeled probes along with tyramide signal amplification (TSA) 3. The commercially available TSA kits allow flexible experimental design as fluorescence emission from green to far-red can be used in combination with various nuclear stains, such as propidium iodide, or fluorescence immunohistochemistry for proteins. TSA produces a reactive fluorescent substrate that quickly covalently binds to moieties, typically tyrosine residues, in the immediate vicinity of the labeled antisense riboprobe. The resulting staining patterns are high resolution in that subcellular localization of the mRNA can be observed using laser scanning confocal microscopy 3,4. One can observe nascent transcripts at the chromosomal loci, distinguish nuclear and cytoplasmic staining and visualize other patterns such as cortical localization of mRNA. Studies in Drosophila indicate that roughly 70% of mRNAs exhibit specific patterns of subcellular localization that frequently correlate with the function of the encoded protein 5. When combined with computer-aided reconstruction of 3D confocal datasets, our protocol allows the detailed analysis of mRNA distribution with sub-cellular resolution in whole vertebrate embryos.

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. We include a propidium iodide nuclear counter-stain to highlight tissue organization.

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology.  Here, we present a high-resolution double ...

Drosophila Pupal Abdomen Immunohistochemistry

The Drosophila pupal abdomen is an established model system for the study of epithelial morphogenesis and the development of sexually dimorphic morphologies 1-3. During pupation, which spans approximately 96 hours (at 25 &deg;C), proliferating populations of imaginal cells replace the larval epidermis to generate the adult abdominal segments. These imaginal cells, born during embryogenesis, exist as lateral pairs of histoblast nests in each abdominal segment of the larvae. Four pairs of histoblast nests give rise to the adult dorsal cuticle (anterior and posterior dorsal nests), the ventral cuticle (ventral nests) and the spiracles associated with each segment (spiracle nests) 4. Upon puparation, these diploid cells (distinguishable by size from the larger polyploid larval epidermal cells- LECs) begin a stereotypical process of proliferation, migration and replacement of the LECs. Various molecular and genetic tools can be employed to investigate the contributions of genetic pathways involved in morphogenesis of the adult abdomen. Ultimate adult phenotypes are typically analyzed following dissection of adult abdominal cuticles. However, investigation of the underlying molecular processes requires immunohistochemical analyses of the pupal epithelium, which present unique challenges. Temporally dynamic morphogenesis and the interactions of two distinct epithelial populations (larval and imaginal) generate a fragile tissue prone to excessive cell loss during dissection and subsequent processing. We have developed methods of dissection, fixation, mounting and imaging of the Drosophila pupal abdominem epithelium for immunohistochemical studies that generate consistent high quality samples suitable for confocal or standard fluorescent microscopy.

Drosophila Pupal Abdomen Immunohistochemistry

Antibody staining of the Drosophila pupae can enhance genetic analyses of adult abdominal developmental genetics. We present our protocol for dissection, fixation and antibody staining of staged Drosophila pupal abdomen.

The Drosophila pupal abdomen is an established model system for the study of epithelial morphogenesis and the development of sexually dimorphic ...

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

Assessing the expression pattern of a gene, as well as the subcellular localization properties of its transcribed RNA, are key features for understanding its biological function during development. RNA in situ hybridization (RNA-ISH) is a powerful method used for visualizing RNA distribution properties, be it at the organismal, cellular or subcellular levels 1. RNA-ISH is based on the hybridization of a labeled nucleic acid probe (e.g. antisense RNA, oligonucleotides) complementary to the sequence of an mRNA or a non-coding RNA target of interest 2. As the procedure requires primary sequence information alone to generate sequence-specific probes, it can be universally applied to a broad range of organisms and tissue specimens 3. Indeed, a number of large-scale ISH studies have been implemented to document gene expression and RNA localization dynamics in various model organisms, which has led to the establishment of important community resources 4-11. While a variety of probe labeling and detection strategies have been developed over the years, the combined usage of fluorescently-labeled detection reagents and enzymatic signal amplification steps offer significant enhancements in the sensitivity and resolution of the procedure 12. Here, we describe an optimized fluorescent in situ hybridization method (FISH) employing tyramide signal amplification (TSA) to visualize RNA expression and localization dynamics in staged Drosophila embryos. The procedure is carried out in 96-well PCR plate format, which greatly facilitates the simultaneous processing of large numbers of samples.

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

Here we describe a whole-mount fluorescent in situ hybridization (FISH) protocol for determining the expression and localization properties of RNAs expressed during embryogenesis in the fruit fly, Drosophila melanogaster.

Assessing  the expression pattern of a gene, as well as the subcellular localization  properties of its transcribed RNA, are key features for ...

Sonication-facilitated Immunofluorescence Staining of Late-stage Embryonic and Larval Drosophila Tissues In Situ

Studies performed in Drosophila melanogaster embryos and larvae provide crucial insight into developmental processes such as cell fate specification and organogenesis. Immunostaining allows for the visualization of developing tissues and organs. However, a protective cuticle that forms at the end of embryogenesis prevents permeation of antibodies into late-stage embryos and larvae. While dissection prior to immunostaining is regularly used to analyze Drosophila larval tissues, it proves inefficient for some analyses because small tissues may be difficult to locate and isolate. Sonication provides an alternative to dissection in larval Drosophila immunostaining protocols. It allows for quick, simultaneous processing of large numbers of late-stage embryos and larvae and maintains in situ morphology. After fixation in formaldehyde, a sample is sonicated. Sample is then subjected to immunostaining with antigen-specific primary antibodies and fluorescently labeled secondary antibodies to visualize target cell types and specific proteins via fluorescence microscopy. During the process of sonication, proper placement of a sonicating probe above the sample, as well as the duration and intensity of sonication, is critical. Additonal minor modifications to standard immunostaining protocols may be required for high quality stains. For antibodies with low signal to noise ratio, longer incubation times are typically necessary. As a proof of concept for this sonication-facilitated protocol, we show immunostains of three tissue types (testes, ovaries, and neural tissues) at a range of developmental stages.

Sonication-facilitated Immunofluorescence Staining of Late-stage Embryonic and Larval Drosophila Tissues In Situ

Immunostaining is an effective technique for visualizing specific cell types and proteins within tissues. By utilizing sonication, the protocol described here alleviates the need to dissect Drosophila melanogaster tissues from late-stage embryos and larvae before immunostaining. We provide an efficient methodology for the immunostaining of formaldehyde-fixed whole mount larvae.

Studies performed in Drosophila melanogaster embryos and larvae provide crucial insight into developmental processes such as cell fate specification and ...

Immunostaining of Whole-Mount Drosophila Testes for 3D Confocal Analysis of Large Spermatocytes

Drosophila testes are a powerful model system for studying biological processes including stem cell biology, nuclear architecture, meiosis and sperm development. However, immunolabeling of the whole Drosophila testis is often associated with significant non-uniformity of staining due to antibody penetration. Squashed preparations only partially overcome the problem since it decreases the 3D quality of the analyses. Herein, we describe a whole-mount protocol using NP40 and heptane during fixation together with immunolabeling in liquid media. It preserves the volume suitable for confocal microscopy together with reproducible and reliable labeling. We show different examples of 3D reconstitution of spermatocyte nuclei from confocal sections. The intra- and inter-testes reproducibility allows 3D quantification and comparison of fluorescence between single cells from different genotypes. We used different components of the intranuclear MINT structure (Mad1-containing Intra Nuclear Territory) as well as two components associated with the nuclear pore complex to illustrate this protocol and its applications on the largest cells of the testis, the S4-S5 spermatocytes.

Immunostaining of Whole-Mount Drosophila Testes for 3D Confocal Analysis of Large Spermatocytes

We present here an improved protocol for the whole-mount preparation and immunostaining of Drosophila testes, suitable for confocal microscopy and also allowing reproducible and reliable labeling. We illustrate this protocol by 3D representation and quantification of colocalization experiments on the large S4-S5 spermatocytes.

Drosophila testes are a powerful model system for studying biological processes including stem cell biology, nuclear architecture, meiosis and sperm ...

Multiplex Detection of Gene Expression in the Intact Drosophila Brain Using Expansion-Assisted Iterative Fluorescence In Situ Hybridization

Understanding gene expression is essential for deciphering cellular functions. However, methods for analyzing the expression of numerous genes in situ within a given tissue remain limited. The EASI-FISH protocol described here has been adapted to detect the expression of dozens of genes in the intact adult Drosophila central nervous system (CNS) using commercially available reagents. This protocol includes a new gel formulation that enhances gel robustness, enabling multiple rounds of hybridization and allowing the embedding of multiple brains per gel. This improvement increases throughput, facilitates optimal comparison of experimental conditions, and reduces reagent costs. Additionally, by employing the GAL4-UAS system for co-detection of green fluorescent protein (GFP), gene expression can be visualized in specific neuronal or glial cell types. Notably, the high resolution achieved through expansion microscopy, combined with the sensitivity of the method, allows for the detection of single RNA transcripts. This approach effectively integrates high image quality with high throughput, making it a powerful tool for studying gene expression throughout the intact fly brain.

Expansion-Assisted Iterative Fluorescence In Situ Hybridization (EASI-FISH) is a robust technique that combines expansion microscopy with fluorescence in situ hybridization (FISH) to detect the expression of multiple genes in thick tissue. This protocol outlines recent advancements in the method and its adaptation for labeling the intact Drosophila central nervous system..

Understanding gene expression is essential for deciphering cellular functions. However, methods for analyzing the expression of numerous genes in situ ...

A Protocol for Immunohistochemistry and RNA In-situ Distribution within Early Drosophila Embryo

Samenvatting

Meer video's verkennen