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>&lt;strong&gt;Supplies&lt;/strong&gt;</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.&amp;nbsp;</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>&lt;strong&gt;Reagent&lt;/strong&gt;</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&amp;nbsp;</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&amp;nbsp;</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>&lt;strong&gt;Equipment&lt;/strong&gt;</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 ...

immunohistochemical-detection-5-methylcytosine-5

Research

JoVE Journal

Developmental Biology

8.9K Views.  BioTime, Inc. Het doel van dit verslag is om te beschrijven van het protocol voor robuuste immunohistochemische detectie van epigenetische markers, 5-methylcytosine (5mC) en 5-hydroxymethylcytosine (5hmC) bij de ontwikkeling en postmitotic muis netvlies.

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

Immunostaining for DNA Modifications: Computational Analysis of Confocal Images

For several decades, 5-methylcytosine (5mC) has been thought to be the only DNA modification with a functional significance in metazoans. The discovery of enzymatic oxidation of 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) as well as detection of N6-methyladenine (6mA) in the DNA of multicellular organisms provided additional degrees of complexity to the epigenetic research. According to a growing body of experimental evidence, these novel DNA modifications may play specific roles in different cellular and developmental processes. Importantly, as some of these marks (e. g. 5hmC, 5fC and 5caC) exhibit tissue- and developmental stage-specific occurrence in vertebrates, immunochemistry represents an important tool allowing assessment of spatial distribution of DNA modifications in different biological contexts. Here the methods for computational analysis of DNA modifications visualized by immunostaining followed by confocal microscopy are described. Specifically, the generation of 2.5 dimension (2.5D) signal intensity plots, signal intensity profiles, quantification of staining intensity in multiple cells and determination of signal colocalization coefficients are shown. Collectively, these techniques may be operational in evaluating the levels and localization of these DNA modifications in the nucleus, contributing to elucidating their biological roles in metazoans.

Newly discovered oxidized forms of 5-methylcytosine (oxi-mCs), 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) may represent distinct DNA modifications with unique functional roles. Here a semi-quantitative workflow for visualization of oxi-mCs' spatial distribution, signal intensity profiling and colocalization is described.

For several decades, 5-methylcytosine (5mC) has been thought to be the only DNA modification with a functional significance in metazoans. The discovery of ...

Genetics

High Sensitivity 5-hydroxymethylcytosine Detection in Balb/C Brain Tissue

DNA hydroxymethylation is a long known modification of DNA, but has recently become a focus in epigenetic research. Mammalian DNA is enzymatically modified at the 5th carbon position of cytosine (C) residues to 5-mC, predominately in the context of CpG dinucleotides. 5-mC is amenable to enzymatic oxidation to 5-hmC by the Tet family of enzymes, which are believed to be involved in development and disease. Currently, the biological role of 5-hmC is not fully understood, but is generating a lot of interest due to its potential as a biomarker. This is due to several groundbreaking studies identifying 5-hydroxymethylcytosine in mouse embryonic stem (ES) and neuronal cells. Research techniques, including bisulfite sequencing methods, are unable to easily distinguish between 5-mC and 5-hmC . A few protocols exist that can measure global amounts of 5-hydroxymethylcytosine in the genome, including liquid chromatography coupled with mass spectrometry analysis or thin layer chromatography of single nucleosides digested from genomic DNA. Antibodies that target 5-hydroxymethylcytosine also exist, which can be used for dot blot analysis, immunofluorescence, or precipitation of hydroxymethylated DNA, but these antibodies do not have single base resolution.In addition, resolution depends on the size of the immunoprecipitated DNA and for microarray experiments, depends on probe design. Since it is unknown exactly where 5-hydroxymethylcytosine exists in the genome or its role in epigenetic regulation, new techniques are required that can identify locus specific hydroxymethylation. The EpiMark 5-hmC and 5-mC Analysis Kit provides a solution for distinguishing between these two modifications at specific loci. The EpiMark 5-hmC and 5-mC Analysis Kit is a simple and robust method for the identification and quantitation of 5-methylcytosine and 5-hydroxymethylcytosine within a specific DNA locus. This enzymatic approach utilizes the differential methylation sensitivity of the isoschizomers MspI and HpaII in a simple 3-step protocol. Genomic DNA of interest is treated with T4-BGT, adding a glucose moeity to 5-hydroxymethylcytosine. This reaction is sequence-independent, therefore all 5-hmC will be glucosylated; unmodified or 5-mC containing DNA will not be affected. This glucosylation is then followed by restriction endonuclease digestion. MspI and HpaII recognize the same sequence (CCGG) but are sensitive to different methylation states. HpaII cleaves only a completely unmodified site: any modification (5-mC, 5-hmC or 5-ghmC) at either cytosine blocks cleavage. MspI recognizes and cleaves 5-mC and 5-hmC, but not 5-ghmC. The third part of the protocol is interrogation of the locus by PCR. As little as 20 ng of input DNA can be used. Amplification of the experimental (glucosylated and digested) and control (mock glucosylated and digested) target DNA with primers flanking a CCGG site of interest (100-200 bp) is performed. If the CpG site contains 5-hydroxymethylcytosine, a band is detected after glucosylation and digestion, but not in the non-glucosylated control reaction. Real time PCR will give an approximation of how much hydroxymethylcytosine is in this particular site. In this experiment, we will analyze the 5-hydroxymethylcytosine amount in a mouse Babl/C brain sample by end point PCR.

The EpiMark 5-hmC and 5-mC Analysis Kit can be used to analyze and quantitate 5-methylcytosine and 5-hydroxymethylcytosine within a spe cific locus. The kit distinguishes 5-mC from 5-hmC by the addition of glucose to the hydroxyl group of 5-hmC via an enzymatic reaction utilizing &beta;-glucosyltransferase (T4-BGT). When 5-hmC occurs In the context of CCGG, this modification converts a cleavable MspI site to a non-cleavable site.

DNA hydroxymethylation is a long known modification of DNA, but has recently become a focus in epigenetic research.   Mammalian DNA is enzymatically ...

Neuroscience

Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo

Angiogenesis is the complex process of new blood vessel formation defined by the sprouting of new blood vessels from a pre-existing vessel network. Angiogenesis plays a key role not only in normal development of organs and tissues, but also in many diseases in which blood vessel formation is dysregulated, such as cancer, blindness and ischemic diseases. In adult life, blood vessels are generally quiescent so angiogenesis is an important target for novel drug development to try and regulate new vessel formation specifically in disease. In order to better understand angiogenesis and to develop appropriate strategies to regulate it, models are required that accurately reflect the different biological steps that are involved. The mouse neonatal retina provides an excellent model of angiogenesis because arteries, veins and capillaries develop to form a vascular plexus during the first week after birth. This model also has the advantage of having a two-dimensional (2D) structure making analysis straightforward compared with the complex 3D anatomy of other vascular networks. By analyzing the retinal vascular plexus at different times after birth, it is possible to observe the various stages of angiogenesis under the microscope. This article demonstrates a straightforward procedure for analyzing the vasculature of a mouse retina using fluorescent staining with isolectin and vascular specific antibodies.

Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo

The neonatal murine retina provides a well characterized physiological model of angiogenesis, which permits investigations of the roles of different genes or drugs that modulate angiogenesis in an in vivo context. Immunofluorescent staining to accurately visualize the vascular plexus is pivotal to the success of these types of studies.

Angiogenesis  is the complex process of new blood vessel formation defined by the sprouting  of new blood vessels from a pre-existing vessel network. ...

Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the developing cerebral cortex, abnormal mitosis of neural progenitors can cause defects in brain size and function. Hence, there is a critical need for tools to understand the mechanisms of neural progenitor mitosis. Cortical development in rodents is an outstanding model for studying this process. Neural progenitor mitosis is commonly examined in fixed brain sections. This protocol will describe in detail an approach for live imaging of mitosis in ex vivo embryonic brain slices. We will describe the critical steps for this procedure, which include: brain extraction, brain embedding, vibratome sectioning of brain slices, staining and culturing of slices, and time-lapse imaging. We will then demonstrate and describe in detail how to perform post-acquisition analysis of mitosis. We include representative results from this assay using the vital dye Syto11, transgenic mice (histone H2B-EGFP and centrin-EGFP), and in utero electroporation (mCherry-&#945;-tubulin). We will discuss how this procedure can be best optimized and how it can be modified for study of genetic regulation of mitosis. Live imaging of mitosis in brain slices is a flexible approach to assess the impact of age, anatomy, and genetic perturbation in a controlled environment, and to generate a large amount of data with high temporal and spatial resolution. Hence this protocol will complement existing tools for analysis of neural progenitor mitosis.

Neural progenitor mitosis is a critical parameter of neurogenesis. Much of our understanding of neural progenitor mitosis is based on analysis of fixed tissue. Live imaging in embryonic brain slices is a versatile technique to assess mitosis with high temporal and spatial resolution in a controlled environment.

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the ...

Rapid and Efficient Spatiotemporal Monitoring of Normal and Aberrant Cytosine Methylation within Intact Zebrafish Embryos

Cytosine methylation is highly conserved across vertebrate species and, as a key driver of epigenetic programming and chromatin state, plays a critical role in early embryonic development. Enzymatic modifications drive active methylation and demethylation of cytosine into 5-methylcytosine (5-mC) and subsequent oxidation of 5-mC into 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine. Epigenetic reprogramming is a critical period during in utero development, and maternal exposure to chemicals has the potential to reprogram the epigenome within offspring. This can potentially cause adverse outcomes such as immediate phenotypic consequences, long-term effects on adult disease susceptibility, and transgenerational effects of inherited epigenetic marks. Although bisulfite-based sequencing enables investigators to interrogate cytosine methylation at base-pair resolution, sequencing-based approaches are cost-prohibitive and, as such, preclude the ability to monitor cytosine methylation across developmental stages, multiple concentrations per chemical, and replicate embryos per treatment. Due to the ease of automated in vivo imaging, genetic manipulations, rapid ex utero development time, and husbandry during embryogenesis, zebrafish embryos continue to be used as a physiologically intact model for uncovering xenobiotic-mediated pathways that contribute to adverse outcomes during early embryonic development. Therefore, using commercially available 5-mC-specific antibodies, we describe a cost-effective strategy for rapid and efficient spatiotemporal monitoring of cytosine methylation within individual, intact zebrafish embryos by leveraging whole-mount immunohistochemistry, automated high-content imaging, and efficient data processing using programming language prior to statistical analysis. To current knowledge, this method is the first to successfully detect and quantify 5-mC levels in situ within zebrafish embryos during early development. The method enables the detection of DNA methylation within the cell mass and also has the ability to detect cytosine methylation of yolk-localized maternal mRNAs during the maternal-to-zygotic transition. Overall, this method will be useful for the rapid identification of chemicals that have the potential to disrupt cytosine methylation in situ during epigenetic reprogramming.

This paper describes a protocol for the rapid and efficient spatiotemporal monitoring of normal and aberrant cytosine methylation within intact zebrafish embryos.

Cytosine methylation is highly conserved across vertebrate species and, as a key driver of epigenetic programming and chromatin state, plays a critical ...

Selective Capture of 5-hydroxymethylcytosine from Genomic DNA

5-methylcytosine (5-mC) constitutes ~2-8% of the total cytosines in human genomic DNA and impacts a broad range of biological functions, including gene expression, maintenance of genome integrity, parental imprinting, X-chromosome inactivation, regulation of development, aging, and cancer1. Recently, the presence of an oxidized 5-mC, 5-hydroxymethylcytosine (5-hmC), was discovered in mammalian cells, in particular in embryonic stem (ES) cells and neuronal cells2-4. 5-hmC is generated by oxidation of 5-mC catalyzed by TET family iron (II)/&alpha;-ketoglutarate-dependent dioxygenases2, 3. 5-hmC is proposed to be involved in the maintenance of embryonic stem (mES) cell, normal hematopoiesis and malignancies, and zygote development2, 5-10. To better understand the function of 5-hmC, a reliable and straightforward sequencing system is essential. Traditional bisulfite sequencing cannot distinguish 5-hmC from 5-mC11. To unravel the biology of 5-hmC, we have developed a highly efficient and selective chemical approach to label and capture 5-hmC, taking advantage of a bacteriophage enzyme that adds a glucose moiety to 5-hmC specifically12.
Here we describe a straightforward two-step procedure for selective chemical labeling of 5-hmC. In the first labeling step, 5-hmC in genomic DNA is labeled with a 6-azide-glucose catalyzed by &beta;-GT, a glucosyltransferase from T4 bacteriophage, in a way that transfers the 6-azide-glucose to 5-hmC from the modified cofactor, UDP-6-N3-Glc (6-N3UDPG). In the second step, biotinylation, a disulfide biotin linker is attached to the azide group by click chemistry. Both steps are highly specific and efficient, leading to complete labeling regardless of the abundance of 5-hmC in genomic regions and giving extremely low background. Following biotinylation of 5-hmC, the 5-hmC-containing DNA fragments are then selectively captured using streptavidin beads in a density-independent manner. The resulting 5-hmC-enriched DNA fragments could be used for downstream analyses, including next-generation sequencing.
Our selective labeling and capture protocol confers high sensitivity, applicable to any source of genomic DNA with variable/diverse 5-hmC abundances. Although the main purpose of this protocol is its downstream application (i.e., next-generation sequencing to map out the 5-hmC distribution in genome), it is compatible with single-molecule, real-time SMRT (DNA) sequencing, which is capable of delivering single-base resolution sequencing of 5-hmC.

Described is a two-step labeling process using &beta;-glucosyltransferase (&beta;-GT) to transfer an azide-glucose to 5-hmC, followed by click chemistry to transfer a biotin linker for easy and density-independent enrichment. This efficient and specific labeling method enables enrichment of 5-hmC with extremely low background and high-throughput epigenomic mapping via next-generation sequencing.

5-methylcytosine (5-mC) constitutes ~2-8% of the total cytosines in human genomic DNA and impacts a broad range of biological functions, including gene ...

Biology

The Detection of 5-Hydroxymethylcytosine in Neural Stem Cells and Brains of Mice

Multiple&#160;DNA modifications have been identified in the mammalian genome. Of that, 5-methylcytosine and 5-hydroxymethylcytosine-mediated epigenetic mechanisms have been intensively studied. 5-hydroxymethylcytosine displays dynamic features during embryonic and postnatal development of the brain, plays a regulatory function in gene expression, and is involved in multiple neurological disorders. Here, we describe the detailed methods including immunofluorescence staining and&#160;DNA dot-blot to detect 5-hydroxymethylcytosine in cultured cells and brain tissues of mouse.

Here, we present a protocol to detect 5-hydroxymethylcytosine in cells and brain tissues, utilizing immunofluorescence staining and DNA dot-blot methods.

Multiple&#160;DNA modifications have been identified in the mammalian genome. Of that, 5-methylcytosine and 5-hydroxymethylcytosine-mediated epigenetic ...

Detection of Modified Forms of Cytosine Using Sensitive Immunohistochemistry

Methylation of cytosine bases (5-methylcytosine, 5mC) occurring in vertebrate genomes is usually associated with transcriptional silencing. 5-hydroxylmethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) are the recently discovered modified cytosine bases produced by enzymatic oxidation of 5mC, whose biological functions remain relatively obscure. A number of approaches ranging from biochemical to antibody based techniques have been employed to study the genomic distribution and global content of these modifications in various biological systems. Although some of these approaches can be useful for quantitative assessment of these modified forms of 5mC, most of these methods do not provide any spatial information regarding the distribution of these DNA modifications in different cell types, required for correct understanding of their functional roles. Here we present a highly sensitive method for immunochemical detection of the modified forms of cytosine. This method permits co-detection of these epigenetic marks with protein lineage markers and can be employed to study their nuclear localization, thus, contributing to deciphering their potential biological roles in different experimental contexts.

Herein we describe a sensitive immunochemical method for mapping the spatial distribution of 5mC oxidation derivatives based on the use of peroxidase-conjugated secondary antibodies and tyramide signal amplification.

Methylation of cytosine bases (5-methylcytosine, 5mC) occurring in vertebrate genomes is usually associated with transcriptional silencing. ...

Cryosectioning and Immunostaining of Mouse Retina

Tissue sectioning and immunohistochemistry are essential techniques in histological and pathological studies of retinal diseases using animal models. These methods enable detailed examinations of tissue morphologies and the localization of specific proteins within the tissue, which provide valuable insights into disease processes and mechanisms. Mice are the most widely used model for this purpose. However, because mouse eyeballs are small and mouse retinas are extremely delicate tissues, obtaining high-quality retinal sections and immunostaining images from mouse eyeballs is typically challenging. This study describes an improved protocol for cryosectioning mouse retinas and performing immunohistochemistry. An essential point of this protocol involves coating the eyeball with a layer of super glue, which prevents deformation of the eyeballs during the processes of cornea removal, lens extraction, and embedding. This step ensures the integrity of retinal morphologies is well preserved. This protocol highlights critical technical considerations and optimization strategies for consistently producing high-quality retinal sections and achieving excellent immunostaining results.

A protocol for preparing mouse retinal cryosections and performing immunostaining on photoreceptors is described. This article enables researchers to consistently produce mouse retinal frozen sections with well-preserved morphology and high-quality immunostaining results.

Tissue sectioning and immunohistochemistry are essential techniques in histological and pathological studies of retinal diseases using animal models. ...

5-Methylcytosine Dot Blot to Determine the Extent of DNA Methylation

Start this procedure by denaturing the isolated DNA in 0.1 molar sodium hydroxide for 10 minutes at 95 degrees Celsius. Neutralize the DNA with 1 molar ammonium acetate on ice, and then dilute two-fold with double-distilled water.
The most critical step in the dot blot is the spotting of the samples onto the membrane, do it slowly, and carefully. Try to minimize each spotted area to 3 to 4 millimeters in diameter.
Using a narrow-mouth pipette tip, carefully spot 2 microliters of the serial diluted genomic DNA onto a positively-charged nylon membrane at the center of the grid. Blot the membrane at 80 degrees Celsius for 30 minutes. Next, block the non-specific antibody binding sites by soaking the membrane in 5% BSA in TBST in a 10-centimeter Petri dish for 1 hour at room temperature with gentle shaking.
After 1 hour, wash the membrane three times in TBST for 5 minutes each time. Incubate the membrane with a mouse anti-5-methylcytosine monoclonal antibody in TBST at 4 degrees Celsius overnight. On the following day, wash the membrane three times in TBST for 5 minutes each time.
Then, incubate with a secondary antibody &#8212; horseradish peroxidase-conjugated sheep anti-mouse immunoglobulin G in TBST for one hour at room temperature. After washing the membrane in TBST as before, add the enzyme substrate to the membrane, and incubate for 5 to 10 minutes. Finally, visualize the secondary antibody signal using a chemiluminescence kit according to the manufacturer&#39;s instructions.

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

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