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>

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M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Remodeling+of+ryanodine+receptor+complex+causes+&amp;#34;leaky&amp;#34;+channels:+a+molecular+mechanism+for+decreased+exercise+capacity.">Remodeling of ryanodine receptor complex causes &amp;#34;leaky&amp;#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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FKBP12+is+a+critical+regulator+of+the+heart+rhythm+and+the+cardiac+voltage-gated+sodium+current+in+mice.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FKBP12+is+the+only+FK506+binding+protein+mediating+T-cell+inhibition+by+the+immunosuppressant+FK506.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ca2++release+channels+join+the+'resolution+revolution'.">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.

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			<td>-80&#176;C Ultra low temperature refrigerator</td>
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			<td>Forma 90 Series</td>
			<td>Used for regent storage</td>
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			<td>Agar</td>
			<td>Sigma-Aldrich</td>
			<td>WXBB6360V</td>
			<td>Preparation of grape juice agar plates</td>
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		<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>In-situ Hybridization</td>
		</tr>
		<tr>
			<td>Bouin&#39;s solution</td>
			<td>Sinopharm Chemical Reagent at Beijing</td>
			<td>69945460</td>
			<td>Drosophila Embryo Embedding</td>
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		<tr>
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			<td>Eppendorf</td>
			<td>540BH07808</td>
			<td>In-situ Hybridization</td>
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			<td>Centrifuge tube</td>
			<td>Denville</td>
			<td>C-2170</td>
			<td>Drosophila Embryo Collection</td>
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			<td>Chrome Alum</td>
			<td>Sinopharm Chemical Reagent at Beijing</td>
			<td>10001018</td>
			<td>Coating Slides</td>
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		<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>
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			<td>Sigma-Aldrich</td>
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			<td>Roche</td>
			<td>11093274910</td>
			<td>RNA labeling with diagoxigenin-UTP by in vitro transcription with SP6 and T7 RNA polymerase</td>
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			<td>Drosophila melanogaster</td>
			<td>Bloomington Stock Center</td>
			<td>BDSC_16799, BDSC_19894, BDSC_11664</td>
			<td>The stocks of Drosophila melanogaster mutant</td>
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		<tr>
			<td>Electric blast drying oven</td>
			<td>Tianjin Taiste Instruments</td>
			<td>101-0AB</td>
			<td>For coating slides and paraffin embedding</td>
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		<tr>
			<td>Eosin</td>
			<td>Sigma-Aldrich</td>
			<td>230251</td>
			<td>Hematoxylin-Eosin Staining</td>
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		<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>
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		<tr>
			<td>Gelatin</td>
			<td>Sinopharm Chemical Reagent at Beijing</td>
			<td>10010328</td>
			<td>Coating Slides</td>
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		<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>
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		<tr>
			<td>LE Agarose</td>
			<td>HyAgarose</td>
			<td>14190108029</td>
			<td>Pre-embedding</td>
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		<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>
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		<tr>
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			<td>Sinopharm Chemical Reagent at Beijing</td>
			<td>10004160</td>
			<td>Hematoxylin-Eosin Staining</td>
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		<tr>
			<td>N-heptane</td>
			<td>Sinopharm Chemical Reagent at Beijing</td>
			<td>40026768</td>
			<td>Drosophila Embryo Collection</td>
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			<td>Paraffin slicer</td>
			<td>Huahai science instrument</td>
			<td>HH-2508III</td>
			<td>In-situ Hybridization</td>
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		<tr>
			<td>Paraffin</td>
			<td>Sinopharm Chemical Reagent at Beijing</td>
			<td>69019461</td>
			<td>Paraffin Embedding</td>
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			<td>pH/mV Meter</td>
			<td>Sartorius</td>
			<td>PB-10</td>
			<td>For determing the pH value of a solution</td>
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			<td>Silver nitrate</td>
			<td>Sinopharm Chemical Reagent at Beijing</td>
			<td>10018461</td>
			<td>Periodic Acid-Silver Methenamine Staining</td>
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		<tr>
			<td>Ultrapure water meter</td>
			<td>Thermo</td>
			<td>AFXI-0501-P</td>
			<td>In-situ Hybridization</td>
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		<tr>
			<td>Xylene</td>
			<td>Sinopharm Chemical Reagent at Beijing</td>
			<td>10023418</td>
			<td>Paraffin Embedding</td>
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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 ...

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Developmental Biology

3.5K Views.  Shaanxi Normal University College of Life Sciences. 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.

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

Understanding Early Organogenesis Using a Simplified In Situ Hybridization Protocol in Xenopus

Organogenesis is the study of how organs are specified and then acquire their specific shape and functions during development. The Xenopuslaevis embryo is very useful for studying organogenesis because their large size makes them very suitable for identifying organs at the earliest steps in organogenesis. At this time, the primary method used for identifying a specific organ or primordium is whole mount in situ hybridization with labeled antisense RNA probes specific to a gene that is expressed in the organ of interest. In addition, it is relatively easy to manipulate genes or signaling pathways in Xenopus and in situ hybridization allows one to then assay for changes in the presence or morphology of a target organ. Whole mount in situ hybridization is a multi-day protocol with many steps involved. Here we provide a simplified protocol with reduced numbers of steps and reagents used that works well for routine assays. In situ hybridization robots have greatly facilitated the process and we detail how and when we utilize that technology in the process. Once an in situ hybridization is complete, capturing the best image of the result can be frustrating. We provide advice on how to optimize imaging of in situ hybridization results. Although the protocol describes assessing organogenesis in Xenopus laevis, the same basic protocol can almost certainly be adapted to Xenopus tropicalis and other model systems.

Understanding Early Organogenesis Using a Simplified In Situ Hybridization Protocol in Xenopus

The Xenopus laevis embryo continues to be exceptionally useful in the study of early development due to its large size and ease of manipulation. A simplified protocol for whole mount in situ hybridization protocol is provided that can be used in the identification of specific organs in this model system.

Organogenesis is the study of how organs are specified and then acquire their specific shape and functions during development.  The Xenopuslaevis embryo ...

Medium-scale Preparation of Drosophila Embryo Extracts for Proteomic Experiments

Analysis of protein-protein interactions (PPIs) has become an indispensable approach to study biological processes and mechanisms, such as cell signaling, organism development, and disease. It is often desirable to obtain PPI information using in vivo material, to gain the most natural and unbiased view of the interaction networks. The fruit fly Drosophila melanogaster is an excellent platform to study PPIs in vivo, and lends itself to straightforward approaches to isolating material for biochemical experiments. In particular, fruit fly embryos represent a convenient type of tissue to study PPIs, due to the ease of collecting animals at this developmental stage and the fact that the majority of proteins are expressed in embryogenesis, thus providing a relevant environment to reveal most PPIs. Here we present a protocol for collection of Drosophila embryos at medium scale (0.5-1 g), which is an ideal amount for a wide range of proteomic applications, including analysis of PPIs by affinity purification-mass spectrometry (AP-MS). We describe our designs for 1 L and 5 L cages for embryo collections that can be easily and inexpensively set up in any laboratory. We also provide a general protocol for embryo collection and protein extraction to generate lysates that can be directly used in downstream applications such as AP-MS. Our goal is to provide an accessible means for all researchers to carry out the analyses of PPIs in vivo.

Medium-scale Preparation of Drosophila Embryo Extracts for Proteomic Experiments

The goal of this protocol is to provide a straightforward and inexpensive approach to collecting Drosophila embryos at medium scale (0.5-1 g) and preparing protein extracts that can be used in downstream proteomic applications, such as affinity purification-mass spectrometry (AP-MS).

Analysis of protein-protein interactions (PPIs) has become an indispensable approach to study biological processes and mechanisms, such as cell signaling, ...

Visualizing Multiciliated Cells in the Zebrafish Through a Combined Protocol of Whole Mount Fluorescent In Situ Hybridization and Immunofluorescence

In recent years, the zebrafish embryo has emerged as a popular model to study developmental biology due to traits such as ex utero embryo development and optical transparency. In particular, the zebrafish embryo has become an important organism to study vertebrate kidney organogenesis as well as multiciliated cell (MCC) development. To visualize MCCs in the embryonic zebrafish kidney, we have developed a combined protocol of whole-mount fluorescent in situ hybridization (FISH) and whole mount immunofluorescence (IF) that enables high resolution imaging. This manuscript describes our technique for co-localizing RNA transcripts and protein as a tool to better understand the regulation of developmental programs through the expression of various lineage factors.

Visualizing Multiciliated Cells in the Zebrafish Through a Combined Protocol of Whole Mount Fluorescent In Situ Hybridization and Immunofluorescence

Cilia development is vital to proper organogenesis. This protocol describes an optimized method to label and visualize ciliated cells of the zebrafish.

In recent years, the zebrafish embryo has emerged as a popular model to study developmental biology due to traits such as ex utero embryo development and ...

Dissection of the Drosophila Pupal Retina for Immunohistochemistry, Western Analysis, and RNA Isolation

The Drosophila pupal retina provides an excellent model system for the study of morphogenetic processes during development. In this paper, we present a reliable protocol for the dissection of the delicate Drosophila pupal retina. Our surgical approach utilizes readily-available microdissection tools to open pupae and precisely extract eye-brain complexes. These can be fixed, subjected to immunohistochemistry, and retinas then mounted onto microscope slides and imaged if the goal is to detect cellular or subcellular structures. Alternatively, unfixed retinas can be isolated from brain tissue, lysed in appropriate buffers and utilized for protein gel electrophoresis or mRNA extraction (to assess protein or gene expression, respectively). Significant practice and patience may be required to master the microdissection protocol described, but once mastered, the protocol enables relatively quick isolation of mainly undamaged retinas.

Dissection of the Drosophila Pupal Retina for Immunohistochemistry, Western Analysis, and RNA Isolation

This paper presents a surgical method for dissecting Drosophila pupal retinas along with protocols for the processing of tissue for immunohistochemistry, western analysis, and RNA-extraction.&#160;

The Drosophila pupal retina provides an excellent model system for the study of morphogenetic processes during development. In this paper, we present a ...

Live Imaging and Analysis of Muscle Contractions in Drosophila Embryo

Coordinated muscle contractions are a form of rhythmic behavior seen early during development in Drosophila embryos. Neuronal sensory feedback circuits are required to control this behavior. Failure to produce the rhythmic pattern of contractions can be indicative of neurological abnormalities. We previously found that defects in protein O-mannosylation, a posttranslational protein modification, affect the axon morphology of sensory neurons and result in abnormal coordinated muscle contractions in embryos. Here, we present a relatively simple method for recording and analyzing the pattern of peristaltic muscle contractions by live imaging of late stage embryos up to the point of hatching, which we used to characterize the muscle contraction phenotype of protein O-mannosyltransferase mutants. Data obtained from these recordings can be used to analyze muscle contraction waves, including frequency, direction of propagation and relative amplitude of muscle contractions at different body segments. We have also examined body posture and taken advantage of a fluorescent marker expressed specifically in muscles to accurately determine the position of the embryo midline. A similar approach can also be utilized to study various other behaviors during development, such as embryo rolling and hatching.

Live Imaging and Analysis of Muscle Contractions in Drosophila Embryo

Here, we present a method to record embryonic muscle contractions in Drosophila embryos in a non-invasive and detail-oriented manner.

Coordinated muscle contractions are a form of rhythmic behavior seen early during development in Drosophila embryos. Neuronal sensory feedback circuits ...

Thawing, Culturing, and Cryopreserving Drosophila Cell Lines

There are currently over 160 distinct Drosophila cell lines distributed by the Drosophila Genomics Resource Center (DGRC). With genome engineering, the number of novel cell lines is expected to increase. The DGRC aims to familiarize researchers with using Drosophila cell lines as an experimental tool to complement and drive their research agenda. Procedures for working with a variety of Drosophila cell lines with distinct characteristics are provided, including protocols for thawing, culturing, and cryopreserving cell lines. Importantly, this publication demonstrates the best practices required to work with Drosophila cell lines to minimize the risk of contaminations from adventitious microorganisms or from other cell lines. Researchers who become familiar with these procedures will be able to delve into the many applications that use Drosophila cultured cells including biochemistry, cell biology and functional genomics.

Thawing, Culturing, and Cryopreserving Drosophila Cell Lines

Drosophila cell lines are important reagents for both fundamental and biomedical research. This article provides protocols for thawing, subculturing, and the cryopreservation of commonly used Drosophila cell lines to assist researchers in incorporating the use of these reagents in their research.

There are currently over 160 distinct Drosophila cell lines distributed by the Drosophila Genomics Resource Center (DGRC). With genome engineering, the ...

A Direct and Simple Method to Assess Drosophila melanogaster's Viability from Embryo to Adult

In Drosophila melanogaster, viability assays are used to determine the fitness of certain genetic backgrounds. Allelic variations can result in partial or complete loss of viability at different stages of development. Our lab has developed a method to assess viability in Drosophila from embryo to fully mature adult. The method relies on quantifying the number of progeny present at different stages during development, starting with hatched embryos. After embryos have been quantified, additional stages are counted, including L1/L2, pupae, and mature adults. After all stages have been examined, a statistical analysis such as the chi-square test is used to determine if there is a significant difference between the starting number of progeny (hatched embryos) and later stages culminating in the observed number of adults, thus rejecting or accepting the null hypothesis (that the number of hatched embryos will be equal to the number of larvae, pupae, and adults recorded throughout the stages of development). The primary advantage of this assay is its simplicity and accuracy, as it does not require an embryo rinse to transfer them to the food vial, avoiding losses from technical errors. Although the protocol described here does not directly examine L2/L3 larvae, additional steps can be added to account for these. Comparing the number of hatched embryos, L1, pupae, and adults can help determine if viability was compromised during the L2/L3 stages for further studies (the use of colored food helps with visual identification of larvae). Overall, this method can help Drosophila researchers and educators determine when viability is compromised during the fly life cycle. Routine assessment of stocks using this assay can prevent accumulation of secondary mutations that may affect the phenotype of the originally isolated mutant, especially if the original mutations affect fitness. For this reason, our lab maintains multiple copies of each of our Dm ime4 alleles and routinely checks the purity of each stock with this method in addition to other molecular analyses.&#160;

A Direct and Simple Method to Assess Drosophila melanogaster's Viability from Embryo to Adult

This protocol is designed to assess the viability of Drosophila at every developmental stage, from embryo to adult. The method can be used to determine and compare the viability of different genotypes or growth conditions.

In Drosophila melanogaster, viability assays are used to determine the fitness of certain genetic backgrounds. Allelic variations can result in partial or ...

Preparation of Small RNA Libraries for Sequencing from Early Mouse Embryos

MicroRNAs (miRNAs) are important for the complex regulation of cell fate decisions and developmental timing. In vivo studies of the contribution of miRNAs during early development are technically challenging due to the limiting cell number. Moreover, many approaches require a miRNA of interest to be defined in assays such as northern blotting, microarray, and qPCR. Therefore, the expression of many miRNAs and their isoforms have not been studied during early development. Here, we demonstrate a protocol for small RNA sequencing of sorted cells from early mouse embryos to enable relatively unbiased profiling of miRNAs in early populations of neural crest cells. We overcome the challenges of low cell input and size selection during library preparation using amplification and gel-based purification. We identify embryonic age as a variable accounting for variation between replicates and stage-matched mouse embryos must be used to accurately profile miRNAs in biological replicates. Our results suggest that this method can be broadly applied to profile the expression of miRNAs from other lineages of cells. In summary, this protocol can be used to study how miRNAs regulate developmental programs in different cell lineages of the early mouse embryo.

We describe a technique for profiling microRNAs in early mouse embryos. This protocol overcomes the challenge of low cell input and small RNA enrichment. This assay can be used to analyze changes in miRNA expression over time in different cell lineages of the early mouse embryo.

MicroRNAs (miRNAs) are important for the complex regulation of cell fate decisions and developmental timing. In vivo studies of the contribution of miRNAs ...

Dissection, Immunohistochemistry and Mounting of Larval and Adult Drosophila Brains for Optic Lobe Visualization

The Drosophila optic lobe, comprised of four neuropils: the lamina, medulla, lobula and lobula plate, is an excellent model system for exploring the developmental mechanisms that generate neural diversity and drive circuit assembly. Given its complex three-dimensional organization, analysis of the optic lobe requires that one understand how its adult neuropils and larval progenitors are positioned relative to each other and the central brain. Here, we describe a protocol for the dissection, immunostaining and mounting of larval and adult brains for optic lobe imaging. Special emphasis is placed on the relationship between mounting orientation and the spatial organization of the optic lobe. We describe three mounting strategies in the larva (anterior, posterior and lateral) and three in the adult (anterior, posterior and horizontal), each of which provide an ideal imaging angle for a distinct optic lobe structure.

Dissection, Immunohistochemistry and Mounting of Larval and Adult Drosophila Brains for Optic Lobe Visualization

This protocol describes three steps to prepare larval and adult Drosophila optic lobes for imaging: 1) brain dissections, 2) immunohistochemistry and 3) mounting. Emphasis is placed on step 3, as distinct mounting orientations are required to visualize specific optic lobe structures.&#160;

The Drosophila optic lobe, comprised of four neuropils: the lamina, medulla, lobula and lobula plate, is an excellent model system for exploring the ...

Live Imaging of Early Cardiac Progenitors in the Mouse Embryo

The first steps of heart development imply drastic changes in cell behavior and differentiation. While analysis of fixed embryos allows studying in detail specific developmental stages in a still snapshot, live imaging captures dynamic morphogenetic events, such as cell migration, shape changes, and differentiation, by imaging the embryo as it develops. This complements fixed analysis and expands the understanding of how organs develop during embryogenesis. Despite its advantages, live imaging is rarely used in mouse models because of its technical challenges. Early mouse embryos are sensitive when cultured ex vivo and require efficient handling. To facilitate a broader use of live imaging in mouse developmental research, this paper presents a detailed protocol for two-photon live microscopy that allows long-term acquisition in mouse embryos. In addition to the protocol, tips are provided on embryo handling and culture optimization. This will help understand key events in early mouse organogenesis, enhancing the understanding of cardiovascular progenitor biology.

We present a detailed protocol for mouse embryo culture and imaging that enables 3D + time imaging of cardiac progenitor cells. This video-toolkit addresses the key skills required for successful live imaging otherwise hard to acquire from text-only publications.

The first steps of heart development imply drastic changes in cell behavior and differentiation. While analysis of fixed embryos allows studying in detail ...