This work was supported by a SBIR grant (1R44EY027654-01A1).

All procedures with mice were performed in accordance with protocols approved by the Children&#39;s Hospital Oakland Research Institute animal care and use committee and adhered to the Association for Research in Vision and Ophthalmology (ARVO) statement for the use of animals in ophthalmic and vision research.
1. Tissue Preparation for Immunostaining
<ol>
	<li>Euthanize C57 BL/6J mice at embryonic day (E) 16, postnatal day (P) 0, P15 and P90 by carbon dioxide (CO2) followed by decapitation (E16 and P0 mice).</li>
	<li>Immediately enucleate mouse eyes punctured with 29G1/2 needle on the dorsal side of the eye. Incubate for 5 minutes&#160;(min) in 1 mL of 4% paraformaldehyde (PFA) in phosphate-buffered saline (1x PBS) pH 8.0 at room temperature.</li>
	<li>To make eye cups from P0, P15 and P90, dissect out cornea and lens with fine ophthalmic scissors while eyes are in the 4% PFA. 
	NOTE: Skip this step for E16 eyes as the eyes are too small.</li>
	<li>Fix the eye cups (P0, P15 and P90) and the whole eyes (E16) in 4% PFA for 20 min&#160;at room temperature. Then wash the eye cups and whole eyes twice in 1 mL of 1x PBS for 10 min at room temperature.</li>
	<li>Cryoprotect the eye cups and whole eyes in 1xPBS, pH 8.0 containing 10% sucrose for 1 hour. Keep in 20% sucrose in 1x PBS for 1 hour and then saturate in 30% sucrose in 1x PBS overnight at 4 &#176;C.</li>
	<li>The next day, embed the eye cups and the whole eyes in optimal cutting temperature compound (OCT), (500 &#181;L) in cryomolds, snap-freeze in a dry ice/ethanol bath, and store at -80 &#176;C.</li>
	<li>Remove the molds with embedded eye cups and whole eyes from -80 &#176;C and allow to equilibrate to -20 &#176;C for 1 hour before cryosectioning.</li>
	<li>Cut retinal cross sections (12 &#181;m thick) parallel to the temporonasal axis through the optic nerve head using a cryostat at -20 &#176;C.</li>
	<li>Mount retinal sections on microscope slides53 and store at -80 &#176;C.</li>
</ol>
2. Immunostaining with 5-mC and 5-hmC Antibodies
<ol>
	<li>Encircle the retinal sections mounted on slides with a hydrophobic barrier pen. The hydrophobic barrier pen reduces the volume of antibody required to stain the tissue.</li>
	<li>Wash the retinal sections once with 200 &#181;L of 1x PBS for 10 min.</li>
	<li>Permeabilize the retinal sections with 200 &#181;L of 0.1% Triton X-100 in 1x PBS (PBS-T) for 10 min&#160;at room temperature.</li>
	<li>Denature retinal sections for 30 min with 200 &#181;L of freshly made 2 N hydrochloric (HCl) acid in 1x PBS in a 37 &#176;C incubator. 
	NOTE: Careful titration of HCl treatment is needed to optimize the 5mC signal.</li>
	<li>Always include biological replicates (3-4) while optimizing 5mC signal54. 
	NOTE: We did antigen retrieval at 4 time points (15 min, 30 min, 1 h and 2 h) and found 30 min incubation is optimal for 5mC signal. Longer incubation with HCl degrades the structure of nuclei leading to inability to localize 5mC signal to the nucleus.</li>
	<li>After denaturation, neutralize retinal sections by adding 100 &#181;L of 0.1 M Tris-HCl (pH 8.3) on retinal sections for 10 min.</li>
	<li>Incubate the sections in 500 &#181;L of blocking solution (5% preimmune normal goat serum in 0.1% Triton X-100 in 1x PBS) for 1 h at room temperature in a humidity chamber.</li>
	<li>After blocking, add anti-5mC; anti-5hmC antibodies diluted 1:500 in blocking solution to the retinal sections.</li>
	<li>Incubate retinal sections overnight at 4 &#176;C in humidity chamber.</li>
	<li>The next day wash, retinal sections 3 times (10-15 min each time) with 0.1% PBS-T and then incubate in blocking solution with the corresponding secondary antibodies (Goat Anti-Rabbit and Goat Anti-Mouse IgG, diluted; 1:1000), containing DAPI ( 4&#39;, 6-diamidino-2-phenylindole) solution (1 &#181;g/mL) at room temperature for 1 h in blocking solution. 
	NOTE: Avoid drying of sections during all stages of immunodetection.</li>
	<li>Wash the slides three time with 1 mL of 0.1% PBS-T.</li>
	<li>After the last wash, mount the sections with aqueous mounting medium under a coverslip and seal with colorless nail polish.</li>
</ol>
3. Confocal Immunohistochemistry
<ol>
	<li>Orient the sections to have the retinal pigment epithelium side of the optic cup always facing one way (e.g., up), for consistency.</li>
	<li>Perform image capture of immunostained retinal sections at 63X (magnification of an objective lens) with 2X or 3X zoom.</li>
	<li>Generate multiple optical sections of each selected image (z-stacks) in all 3 channels (Red, Green and Blue, RGB) using 0.35 &#181;m step. 
	NOTE: The final magnification of a specimen visualized at 10-20X (magnification of an objective lens) will be 63X, respectively, when combined with a (typically used) 10X ocular lens. 
	Visualize compressed optical stack to locate the 5mC and 5hmC signal</li>
</ol>

The authors have nothing to disclose.

DNA methylation and hydroxymethylation are dynamic and reversible DNA modifications, which modulatethe diverse range of biological mechanisms in a developing as well as in postmitotic cells. Here, we describe a reproducible technique for detecting 5mC and 5hmC DNA modifications in situ by immunohistochemical technique (using anti-5mC and anti-5hmC antibodies) in paraformaldehyde-fixed frozen sections of mouse retina, and provide guidelines for improving and standardizing the results, especially when comparing several different specimens. We earlier used this method to delineate 5mC changes in mouse retina with retina-specific targeting of Dnmt1, Dnmt3a and Dnmt3b DNA methyltransferase genes, and reported the (expected) depletion of 5mC marks in their retinas7.
The critical step in 5mC immunohistochemical detection is optimization of treatment of histological sections with HCl: less than 15 min pretreatment is not expected to produce reproducible results, whereas excessive treatment with HCl destroys the nuclear architecture. We found best result after 30 min incubation with HCl. We recommend to always use freshly diluted HCl and freshly made 4% PFA solution for DNA denaturation and retinal tissue fixation.
To troubleshoot the results, we recommend to include three to four biological replicates while optimizing 5mC signal. While each individual set of immunohistochemical staining may generate slightly different results (more or less bright heterochromatic regions), which is expected in immunohistochemical method, the 5mC and 5hmC nuclear distribution within the individual set of sections is expected to be very reproducible, for as long as the protocol is followed.
This technique also has limitation as we consistently observed variability in 5mC distribution in the photoreceptor cell nuclei within the same section. We found some nuclei showed strong 5mC signal while adjacent cell nuclei showed no 5mC signal. This is due to limitation in unmasking 5mC antigen by HCl treatment in frozen sections.
The significance of this technique enables 5mC localization without DNase and proteinase K treatment leading to better preservation of chromocenters. This technique is expected to work robustly on any sections from all vertebrate animal tissues, carrying 5mC, 5hmC marks, and processed with a similar approach, not only mouse retina, for as long as the protocol is followed.

Epigenetic regulation of development and postmitotic homeostasis of mouse retina is an exciting area of research, which promises to bring a novel understanding of biological mechanisms controlling retinogenesis and cell fate determination, cell type-specific metabolic function as well as cell pathologies, cell death and regeneration1,2,3,4,5,6,7,8,9,10,11,12,13. Chromatin undergoes dynamic changes in development, as well as in response to variable external signals whether it is stress, metabolic or cell death stimuli6,14,15,16,17,18. In mouse nuclei, chromatin is divided into active euchromatin and inactive heterochromatin6,19,20. In interphase nucleus, euchromatin resides in the inner region of the nucleus whereas heterochromatin lines the nuclear periphery and nucleolus. This pattern is called conventional and is highly conserved in eukaryotes. In contrast, rod nuclei in nocturnal animals such as mouse and rat have inverted pattern where the nucleus is occupied by the heterochromatin in the center surrounded by euchromatin forming the outermost shell6,18,20. At birth, rod nuclei have conventional nuclear architecture. Inversion of rod nuclei occurs during development by remodeling of conventional nuclear architecture. During this process, peripheral heterochromatin separates from the nuclear periphery and chromocenters fuse together to form a single chromocenter in the center of the nucleus6,18.
Genomic DNA methylation participates in controlling local chromatin conformation21. DNA methylation occurs at the 5&#39; position of cytosine residues of CpG dinucleotides that are enriched in the promoter region of many genes in mouse genomes22,23,24,25,26 as well as in intergenic and intron regions, which carry regulatory elements27. Methylation of CpG islands in proximal promoters21,24 and CpG sequences in gene enhancers27,28 is important in regulating the dynamic state of bivalent chromatin, containing many developmental genes/transcription factors playing important role in development, cancer and aging29,30,31,32,33,34. Three DNA methyltransferases (DNMTs) participate in DNA methylation during mouse development35. All three DNMTs are expressed during mouse retinal development36 and retina-specific changes in Dnmt genes impact retinal development2,7. Hydroxymethylcytosine (5hmC) is the oxidative product of 5-methylcytosine (5mC) demethylation. The three Ten-Eleven Translocation (TET) enzymes participate in 5mC demethylation37,38,39. Hydroxymethyl-C modification is enriched in promoters, gene bodies and intergenic genomic sequences within gene rich and actively transcribed regions27,40.
There is a variety of described approaches for determining changing DNA methylation patterns in cells41,42,43,44,45,46, some of them enabling quantifying global changes of DNA methylation while others focusing on precise changes in CpG-rich regions within promoters and enhancers. Methods for detecting and quantification of 5hmC were also reported47, including by immunohistochemistry13. Immunohistochemical techniques, which enable robust monitoring of 5mC and 5hmC changes in nuclei of developing and postmitotic cells, are very informative for delineating chromatin dynamics in situ in response to external or internal changes impacting the cells4,13,48,49. However, this approach is prone to underestimating DNA methylation and hydroxymethylation changes due to technical difficulties, associated with detecting cytosine modifications in histological preparations. This is because the nonpolar DNA bases, including 5mC and 5hmC-modified cytosine, are hidden within the center of the double-stranded DNA helix50, and require unmasking. This is especially challenging in frozen histological preparations, which may rapidly lose informative organization of the original tissue when harsh treatment is applied.
The mouse retina is an excellent model to dissect the contribution of DNA methylation to neural development and postmitotic homeostasis. There are only 6 types of neurons (rod and cone photoreceptors, amacrine, bipolar, horizontal and ganglion cells), one glial cell type (Muller glia) and one neuroepithelial cell type (retinal pigment epithelium)51. Retinal cell types were reported to have distinct patterns of DNA methylation18,19, which recently has been studied at single-base resolution1,5,9,12. We recently reported immunohistochemistry application to delineating 5mC pattern of distribution within nuclei of retinal cells and changes in the nuclei of adult mouse retina, where all three DNA methyltransferases have been removed by conditional targeting7. Compared to a number of reports, where the presence of DNA methylation in retinal cells could be viewed only as a positive or negative signal in hypermethylated cells undergoing apoptosis, our approach enabled detecting specific chromatin arrangement within the retinal cell nuclei, which we and several groups reported and discussed earlier5,6,7,18,19,36,52. Here, we describe the immunohistochemical (IHC) technique of detecting 5mC and 5hmC in frozen paraformaldehyde-fixed histological sections in details as well as demonstrate the data, which we were able to reproduce from our original report7.

<table>
	<tbody>
		<tr>
			<td>Supplies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>29G1/2 ULTRA-FINE</td>
			<td>BD Biosciences</td>
			<td>329461</td>
			<td></td>
		</tr>
		<tr>
			<td>Ophthalmic scissor</td>
			<td>Fine Science tools, Inc.&#160;</td>
			<td>15000-03</td>
			<td></td>
		</tr>
		<tr>
			<td>PAP pen</td>
			<td>RPICORP.com</td>
			<td>195505</td>
			<td></td>
		</tr>
		<tr>
			<td>Microslide Superfrost plus</td>
			<td>VWR</td>
			<td>48311-703</td>
			<td></td>
		</tr>
		<tr>
			<td>Reagent</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Electron Microscope Science</td>
			<td>15710</td>
			<td></td>
		</tr>
		<tr>
			<td>1x PBS</td>
			<td>Corning</td>
			<td>21-031-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma</td>
			<td>S9378</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue-Tek Optical cutting compound (OCT)</td>
			<td>VWR</td>
			<td>4583</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X 100</td>
			<td>Sigma</td>
			<td>T9284</td>
			<td></td>
		</tr>
		<tr>
			<td>6 N HCl</td>
			<td>VWR analytical&#160;</td>
			<td>BDH7204-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-HCl pH 8.3</td>
			<td>Teknova</td>
			<td>T1083</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat serum</td>
			<td>Jackson Immunoresearch</td>
			<td>005-000-121</td>
			<td></td>
		</tr>
		<tr>
			<td>5mC</td>
			<td>Genway Biotech&#160;</td>
			<td>GWB-BD5190</td>
			<td></td>
		</tr>
		<tr>
			<td>5hmC</td>
			<td>Active Motif</td>
			<td>39791</td>
			<td></td>
		</tr>
		<tr>
			<td>LaminB1</td>
			<td>Abcam</td>
			<td>Ab16048</td>
			<td></td>
		</tr>
		<tr>
			<td>Cy2 AffiniPure Goat Ant-Rabbit IgG</td>
			<td>Jackson Immunoresearch</td>
			<td>111-225-1444</td>
			<td></td>
		</tr>
		<tr>
			<td>Cy3 AffiniPure Goat Ant-Mouse IgG</td>
			<td>Jackson Immunoresearch</td>
			<td>115-585-166</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Thermo Fisher Scientific</td>
			<td>D1306</td>
			<td></td>
		</tr>
		<tr>
			<td>Prolong Gold Antifade medium</td>
			<td>Thermo Fisher Scientific</td>
			<td>P36930</td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MICRM HM 550</td>
			<td>Thermo Fisher Scientific</td>
			<td>388114</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

immunohistochemical detection of 5-methylcytosine and 5-hydroxymethylcytosine in developing and postmitotic mouse retina

The epigenetics of retinal development is a well-studied research field, which promises to bring a new level of understanding about the mechanisms of a variety of human retinal degenerative diseases and pinpoint new treatment approaches. The nuclear architecture of mouse retina is organized in two different patterns: conventional and inverted. Conventional pattern is universal where heterochromatin is localized to the periphery of the nucleus, while active euchromatin resides in the nuclear interior. In contrast, inverted nuclear pattern is unique to the adult rod photoreceptor cell nuclei where heterochromatin localizes to the nuclear center, and euchromatin resides in the nuclear periphery. DNA methylation is predominantly observed in chromocenters. DNA methylation is a dynamic covalent modification on the cytosine residues (5-methylcytosine, 5mC) of CpG dinucleotides that are enriched in the promoter regions of many genes. Three DNA methyltransferases (DNMT1, DNMT3A and DNMT3B) participate in methylation of DNA during development. Detecting 5mC with immunohistochemical techniques is very challenging, contributing to variability in results, as all DNA bases including 5mC modified bases are hidden within the double-stranded DNA helix. However, detailed delineation of 5mC distribution during development is very informative. Here, we describe a reproducible technique for robust immunohistochemical detection of 5mC and another epigenetic DNA marker 5-hydroxymethylcytosine (5hmC), which colocalizes with the &#34;open&#34;, transcriptionally active chromatin in developing and postmitotic mouse retina.

To determine the distribution of 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) during retinal development and in postmitotic retina, we immunostained C57BL/6J mouse retinal section from E16, P0, P15 and P90 with antibodies specific to 5mC and 5hmC.
At E16 the 5mC staining was strong in chromocenters of the cell nuclei while weak staining was observed in the whole cell nuclei (Figure 1a). The 5hmC staining was confined to the cell nuclei and was absent from the chromocenters (Figure 1b).
In P0 retina, the 5mC signal was localized to the chromocenters of the cell nuclei and nuclear periphery (Figure 2a, arrow and arrowhead). We also observed weak staining of 5mC between the chromocenters of the cell nuclei. The 5hmC signal was strongly present in the cell nuclei except for the chromocenters (Figure 2b). We found more nuclei are stained with 5mC and 5hmC in inner neuroblast layer (INBL) (Figure 2c, dotted line)
In P15 retina, we found strong staining of 5mC and 5hmC in outer nuclear layer (ONL), inner nuclear layer (INL) and ganglion cell layer (GCL) (Figure 3). In ONL (rod photoreceptors), the 5mC signal was present in the chromocenters of the cell nuclei and nuclear periphery (Figures 3a-3c, 3g). The 5hmC signal was found in the whole cell nuclei except for the chromocenters (Figures 3d-3f). The transmission electron microscopy done on P15 retina shows distribution of heterochromatic domains in rod cell nuclei similar to that found with 5mC antibody signal (Figures 3h and 3i).
The 5mC staining in INL and GCL was strong in the chromocenters of the cell nuclei and weak staining was also observed in whole cell nuclei (Figures 3j-3l and 3r-3t). In contrast to 5mC, the 5hmC signal was localized to the whole cell nuclei except for the chromocenters in INL and GCL (Figures 3m-3o and 3u-3w). We observed strong 5hmC signal on the periphery of GCL cell nuclei.
In adult rod photoreceptors, the 5mC signal was restricted to the chromocenter and nuclear periphery of the cell nuclei (Figures 4a-4c) while 5hmc signal was confined to the nuclear periphery (Figures 4d-4f).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58274/58274fig1.jpg" /> 
Figure 1. Localization of 5-methylcytosine and 5-hydroxymethylcytosine in mouse retina at embryonic day 16. 
Immunostaining of C57BL/6J retina with antibodies to 5mC (red, a) and 5hmC (green, b). At E16, 5mC signal is very strong in chromocenters of the cell nuclei and also present in the whole cell nuclei at much lower level. 5hmC staining was exclusively confined to the cell nuclei. Panel c represents merged 5mC and 5hmC image. Nuclei are counterstained with DAPI (blue, c). Insets represent magnification of area shown with asterisk in the images. Scale bar: 5 &#181;m. <a href="https://www.jove.com/files/ftp_upload/58274/58274fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58274/58274fig2.jpg" /> 
Figure 2. Immunohistochemical staining of 5-methylcytosine and 5-hydroxymethylcytosine in mouse retina at postnatal day 0. 
Immunolabeling of C57BL/6J retina with antibodies to 5mC (red, a) and 5hmC (green, b). In P0 retina, 5mC and 5hmC are present in both outer neuroblast layer (ONBL) and inner neuroblast layer (INBL). a) 5mC signal is strongly present in the chromocenters (arrow) and nuclear periphery of the cell nuclei (arrowhead). Weak staining of 5mC is also observed in between the chromocenters of cell nuclei. b) 5hmC staining is confined to the cell nucleus and strong signal is observed in INBL (dotted line in panel c). Panel c represents merged 5mC and 5hmC image. Nuclei are counterstained with DAPI (blue, c). Insets represent magnification of area shown with asterisk in the images. Scale bar: 5 &#181;m. <a href="https://www.jove.com/files/ftp_upload/58274/58274fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58274/58274fig3.jpg" /> 
Figure 3. 5-methylcytosine and 5-hydroxymethylcytosine distribution in mouse retina at postnatal day 15. 
Immunostaining of C57BL/6J retina with anti-5mC and 5hmC antibodies (a-g, j-y). In outer nuclear layer (ONL) (rod photoreceptors), 5mC signal (red, a) usually coincides with the chromocenters of the cell nuclei (arrowhead, b) and also localizes to the nuclear periphery (arrow, b). Panel b (red) and c (grey) represent magnification of area shown with asterisk in the image a. The 5hmC signal (green, d) is confined to the whole cell nuclei except for the chromocenters. Panel e (green) and f (grey) represent magnification of area shown with asterisk in the image d. Panel g represents merged 5mC and 5hmC image. Nuclei are counterstained with DAPI. Panel h and i are transmission electron microscope image of photoreceptor nuclei at postnatal day 15 showing distribution of heterochromatic domains. Black arrowhead in panel h points to single rod photoreceptor nucleus enlarged in panel i. Red arrowheads in panel i point to heterochromatic domains within rod photoreceptor nucleus while red arrows point to heterochromatin in the nuclear periphery. In INL (j-q) and GCL cell nuclei (r-y), the 5mC signal (red) is localized to the chromocenters present in the periphery and weak staining is also detected in the rest of the cell nuclei. Panel k (red) and i (grey) represent magnification of area shown with asterisk in the image j. The 5hmC staining (green) in INL and GCL cell nuclei is confined to the whole cell nucleus. Note strong 5hmC staining in the nuclear periphery of GCL cells. Panel p and x represent merged 5mC and 5hmC image while panel q and y represent magnification of area shown with asterisk in the image p and x respectively. Nuclei are counterstained with DAPI. Scale bar: 5 &#181;m. <a href="https://www.jove.com/files/ftp_upload/58274/58274fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58274/58274fig4.jpg" /> 
Figure 4. 5-methylcytosine and 5-hydroxymethylcytosine distribution in rod photoreceptor nuclei of C57BL/6J mouse retina at postnatal day 90. 
The 5mC staining (red, a) is strongly present in the chromocenter of the cell nuclei and also weakly present in the nuclear periphery. Panel b (red) and c (grey) represent magnification of area shown with asterisk in the image a. The 5hmC staining (green, d) is exclusively confined to the nuclear periphery Panel e (green) and f (grey) represent magnification of area shown with asterisk in the image d. Panel g represents merged 5mC and 5hmC image while panel h is magnification of area shown with asterisk in the image g. Nuclei are counterstained with DAPI (blue). Scale bar: 5 &#181;m. <a href="https://www.jove.com/files/ftp_upload/58274/58274fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Immunohistochemical Detection of 5-Methylcytosine and 5-Hydroxymethylcytosine in Developing and Postmitotic Mouse Retina at JoVE.com

Immunohistochemical Detection of 5-Methylcytosine and 5-Hydroxymethylcytosine in Developing and Postmitotic Mouse Retina

The objective of this report is to describe the protocol for robust immunohistochemical detection of epigenetic markers, 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) in developing and postmitotic mouse retina.

The epigenetics of retinal development is a well-studied research field, which promises to bring a new level of understanding about the mechanisms of a ...

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8.8K Views.  BioTime, Inc. The objective of this report is to describe the protocol for robust immunohistochemical detection of epigenetic markers, 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) in developing and postmitotic mouse retina.

Video: Immunohistochemical Detection of 5-Methylcytosine and 5-Hydroxymethylcytosine in Developing and Postmitotic Mouse Retina

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We present a method to investigate the subcellular protein localization in the larval zebrafish retina by combining super-resolution light microscopy and ...

A Novel Use of Three-dimensional High-frequency Ultrasonography for Early Pregnancy Characterization in the Mouse

High-frequency ultrasonography (HFUS) is a common method to non-invasively monitor the real-time development of the human fetus in utero. The mouse is routinely used as an in vivo model to study embryo implantation and pregnancy progression. Unfortunately, such murine studies require pregnancy interruption to enable follow-up phenotypic analysis. To address this issue, we used three-dimensional (3-D) reconstruction of HFUS imaging data for early detection and characterization of murine embryo implantation sites and their individual developmental progression in utero. Combining HFUS imaging with 3-D reconstruction and modeling, we were able to accurately quantify embryo implantation site number as well as monitor developmental progression in pregnant C57BL6J/129S mice from 5.5 days post coitus (d.p.c.) through to 9.5 d.p.c. with the use of a transducer. Measurements included: number, location, and volume of implantation sites as well as inter-implantation site spacing; embryo viability was assessed by cardiac activity monitoring. In the immediate post-implantation period (5.5 to 8.5 d.p.c.), 3-D reconstruction of the gravid uterus in both mesh and solid overlay format enabled visual representation of the developing pregnancies within each uterine horn. As genetically engineered mice continue to be used to characterize female reproductive phenotypes derived from uterine dysfunction, this method offers a new approach to detect, quantify, and characterize early implantation events in vivo. This novel use of 3-D HFUS imaging demonstrates the ability to successfully detect, visualize, and characterize embryo-implantation sites during early murine pregnancy in a non-invasive manner. The technology offers a significant improvement over current methods, which rely on the interruption of pregnancies for gross tissue and histopathologic characterization. Here we use a video and text format to describe how to successfully perform ultrasounds of early murine pregnancy to generate reliable and reproducible data with reconstruction of the uterine form in mesh and solid 3-D images.

Mice are widely used to study gestational biology. However, pregnancy termination is required for such studies which precludes longitudinal investigations and necessitates the use of large numbers of animals. Therefore, we describe a non-invasive technique of high-frequency ultrasonography for early detection and monitoring of post-implantation events in the pregnant mouse.

High-frequency ultrasonography (HFUS) is a common method to non-invasively monitor the real-time development of the human fetus in utero. The mouse is ...

Dissection and Coronal Slice Preparation of Developing Mouse Pituitary Gland

The pituitary gland or hypophysis is an important endocrine organ secreting hormones essential for homeostasis. It consists of two glands with separate embryonic origins and functions &#8212; the neurohypophysis and the adenohypophysis. The developing mouse pituitary gland is tiny and delicate with an elongated oval shape. A coronal section is preferred to display both the adenohypophysis and neurohypophysis in a single slice of the mouse pituitary.
The goal of this protocol is to achieve proper pituitary coronal sections with well-preserved tissue architectures from developing mice. In this protocol, we describe in detail how to dissect and process pituitary glands properly from developing mice. First, mice are fixed by transcardial perfusion of formaldehyde prior to dissection. Then three different dissecting techniques are applied to obtain intact pituitary glands depending on the age of mice. For fetal mice aged embryonic days (E) 17.5 - 18.5 and neonates up to 4 days, the entire sella regions including the sphenoid bone, gland, and trigeminal nerves are dissected. For pups aged postnatal days (P) 5 - 14, the pituitary glands connected with trigeminal nerves are dissected as a whole. For mice over 3 weeks old, the pituitary glands are carefully dissected free from the surrounding tissues. We also display how to embed the pituitary glands in a proper orientation by using the surrounding tissues as landmarks to obtain satisfying coronal sections. These methods are useful in analyzing histological and developmental features of pituitary glands in developing mice.

We present a protocol to dissect pituitary glands and prepare pituitary coronal sections from developing mice.

The pituitary gland or hypophysis is an important endocrine organ secreting hormones essential for homeostasis. It consists of two glands with separate ...

Preparing Developing Peripheral Olfactory Tissue for Molecular and Immunohistochemical Analysis in Drosophila

The olfactory system of Drosophila is a widely used system in developmental neurobiology, systems neuroscience, as well as neurophysiology, behavior, and behavioral evolution. Drosophila olfactory tissues house the olfactory receptor neurons (ORNs) that detect volatile chemical cues in addition to hydro- and thermo-sensory neurons. In this protocol, we describe the dissection of developing peripheral olfactory tissue of the adult Drosophila species. We first describe how to stage and age Drosophila larvae, followed by the dissection of the antennal disc from early pupal stages, followed by the dissection of the antennae from mid-pupal stages and adults. We also show methods where preparations can be utilized in molecular techniques, such as the RNA extraction for qRT-PCR, RNAseq, or immunohistochemistry. These methods can also be applied to other Drosophila species after species-specific pupal development times are determined, and respective stages are calculated for appropriate aging.

Preparing Developing Peripheral Olfactory Tissue for Molecular and Immunohistochemical Analysis in Drosophila

Here, we present a protocol to stage and dissect developing olfactory tissue from Drosophila species. The dissected tissue can later be used for molecular analyses, such as quantitative RT-PCR (Reverse Transcription-Polymerase Chain Reaction) or RNA sequencing (RNAseq), as well as in vivo analyses such as immunohistochemistry or in situ hybridization.

The olfactory system of Drosophila is a widely used system in developmental neurobiology, systems neuroscience, as well as neurophysiology, behavior, and ...

Tracing Gene Expression Through Detection of &#946;-galactosidase Activity in Whole Mouse Embryos

The Escherichia coli LacZ gene, encoding &#946;-galactosidase, is largely used as a reporter for gene expression and as a tracer in cell lineage studies. The classical histochemical reaction is based on the hydrolysis of the substrate X-gal in combination with ferric and ferrous ions, which produces an insoluble blue precipitate that is easy to visualize. Therefore, &#946;-galactosidase activity serves as a marker for the expression pattern of the gene of interest as the development proceeds. Here we describe the standard protocol for the detection of &#946;-galactosidase activity in early whole mouse embryos and the subsequent method for paraffin sectioning and counterstaining. Additionally, a procedure for clarifying whole embryos is provided to better visualize X-gal staining in deeper regions of the embryo. Consistent results are obtained by performing this procedure, although optimization of reaction conditions is needed to minimize background activity. Limitations in the assay should be also considered, particularly regarding the size of the embryo in whole mount staining. Our protocol provides a sensitive and a reliable method for &#946;-galactosidase detection during the mouse development that can be further applied to the cryostat sections as well as whole organs. Thus, the dynamic gene expression patterns throughout development can be easily analyzed by using this protocol in whole embryos, but also detailed expression at the cellular level can be assessed after paraffin sectioning.

Here we describe the standard protocol for the detection of &#946;-galactosidase activity in early whole mouse embryos and the method for paraffin sectioning and counterstaining. This is an easy and quick procedure to monitor gene expression during development that can also be applied to tissue sections, organs or cultured cells.

The Escherichia coli LacZ gene, encoding &#946;-galactosidase, is largely used as a reporter for gene expression and as a tracer in cell lineage studies. ...

Visualizing the Node and Notochordal Plate In Gastrulating Mouse Embryos Using Scanning Electron Microscopy and Whole Mount Immunofluorescence

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is the formation of the transient organizers, the node and notochordal plate, from cells that have passed through the primitive streak. The proper formation of these signaling centers is essential for the development of the body plan and techniques to visualize them are of high interest to mouse developmental biologists. The node and notochordal plate lie on the ventral surface of gastrulating mouse embryos around embryonic day (E) 7.5 of development. The node is a cup-shaped structure whose cells possess a single slender cilium each. The proper subcellular localization and rotation of the cilia in the node pit determines left-right asymmetry. The notochordal plate cells also possess single cilia albeit shorter than those of the node cells. The notochordal plate forms the notochord which acts as an important signaling organizer for somitogenesis and neural patterning. Because the cells of the node and notochordal plate are transiently present on the surface and possess cilia, they can be visualized using scanning electron microscopy (SEM). Among other techniques used to visualize these structures at the cellular level is whole mount immunofluorescence (WMIF) using the antibodies against the proteins that are highly expressed in the node and notochordal plate. In this report, we describe our optimized protocols to perform SEM and WMIF of the node and notochordal plate in developing mouse embryos to help in the assessment of tissue shape and cellular organization in wild-type and gastrulation mutant embryos.

The node and notochordal plate are transient signaling organizers in developing mouse embryos that can be visualized using several techniques. Here, we describe in detail how to perform two of the techniques to study their structure and morphogenesis: 1) scanning electron microscopy (SEM); and 2) whole mount immunofluorescence (WMIF).

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is ...

Visualization of Tangential Cell Migration in the Developing Chick Optic Tectum

Time-lapse imaging is a powerful method to analyze migrating cell behavior. After fluorescent cell labeling, the movement of the labeled cells in culture can be recorded under video microscopy. For analyzing cell migration in the developing brain, slice culture is commonly used to observe cell migration parallel to the slice section, such as radial cell migration. However, limited information can be obtained from the slice culture method to analyze cell migration perpendicular to the slice section, such as tangential cell migration. Here, we present the protocols for time-lapse imaging to visualize tangential cell migration in the developing chick optic tectum. A combination of cell labeling by electroporation in ovo and a subsequent flat-mount culture on the cell culture insert enables detection of migrating cell movement in the horizontal plane. Moreover, our method facilitates detection of both individual cell behavior and the collective action of a group of cells in the long term. This method can potentially be applied to detect the sequential change of the fluorescent-labeled micro-structure, including the axonal elongation in the neural tissue or cell displacement in the non-neural tissue.

We describe the methods for fluorescent labeling of tangentially migrating cells by electroporation, and for time-lapse imaging of the labeled cell movement in a flat-mount culture in order to visualize migrating cell behavior in the developing chick optic tectum.

Time-lapse imaging is a powerful method to analyze migrating cell behavior. After fluorescent cell labeling, the movement of the labeled cells in culture ...

Assessment of Ultrastructural Neuroplasticity Parameters After In Utero Transduction of the Developing Mouse Brain and Spinal Cord

The present study combines in utero transduction with transmission electron microscopy (TEM) aiming at a precise morphometrical analysis of ultrastructural parameters in unambiguously identified topographical structures, affected by a protein of interest that is introduced into the organism via viral transfer. This combined approach allows for a smooth transition from macrostructural to ultrastructural identification by following topographical navigation maps in a tissue atlas. High-resolution electron microscopy of the in-utero-transduced tissue reveals the fine ultrastructure of the neuropil and its plasticity parameters, such as cross-sectioned synaptic bouton areas, the number of synaptic vesicles and mitochondria within a bouton profile, the length of synaptic contacts, cross-sectioned axonal areas, the thickness of myelin sheaths, the number of myelin lamellae, and cross-sectioned areas of mitochondria profiles. The analysis of these parameters reveals essential insights into changes of ultrastructural plasticity in the areas of the nervous system that are affected by the viral transfer of the genetic construct. This combined method can not only be used for studying the direct effect of genetically engineered biomolecules and/or drugs on neuronal plasticity but also opens the possibility to study the in utero rescue of neuronal plasticity (e.g., in the context of neurodegenerative diseases).

The combination of transmission electron microscopy and in utero transduction is a powerful approach for studying morphological changes in the fine ultrastructure of the nervous system during development. This combined method allows deep insights into changes in structural details underlying neuroplasticity with respect to their topographical representation.