This method can help answer key questions in the DNA methylation field, such as changes in chromatin during retinal development in postmitotic retina. The main advantage of this technique is that it can be used robustly on any sections from all mouse retina tissues carrying 5-methylcytosine, 5-hydroxymethylcytosine distribution that are processed in a similar approach. Visual demonstration of this method is critical as the tissue preparing and staining steps are difficult to learn, because if frozen histological preps are being used, valuable organizational and original tissue can be lost.

Demonstrating the rest of the procedure will be Pablo Diaz, research associate from our laboratory. To start this procedure, use a 29 gauge one half inch needle to puncture the eyes of a decapitated euthanized mouse on the dorsal side of the eyes. To obtain eye cups, immediately enucleate the eyes by cutting around the sclera with fine ophthalmic scissors to remove the cornea and the lens.

Fix the eye cups and E16 whole eyes in 4%paraformaldehyde or PFA in 1xPBS pH 8.0 for 20 minutes at room temperature. Wash them twice in one milliliter of 1xPBS at room temperature for 10 minutes. To cryoprotect the eye cups and whole eyes, incubate them in 1xPBS pH 8.0 with 10%sucrose at room temperature for one hour.

Then in 20%sucrose in 1xPBS for one hour. And finally in 30%sucrose in 1xPBS at 4 degrees Celsius overnight. On the following day, embed the eye cups and eyes in optimal cutting temperature compound, or OCT, in cryomolds.

Then snap freeze them in a dry ice ethanol bath and keep at 80 degrees Celsius. Prior to starting cryosectioning, remove the molds with embedded eye cups and eyes from 80 degrees Celsius. Place them in a cryostat holder and allow them to equilibrate to 20 degrees Celsius for one hour.

Using a cryostat at 20 degrees Celsius, cut 12 micrometer thick retinal cross sections parallel to the temporonasal axis through the optic nerve head. Mount the sections on microscope slides and store at 80 degrees Celsius. To start immunostaining, use a hydrophobic barrier pen to encircle the retinal sections mounted on slides.

Wash the sections once with 200 microliters of 1xPBS for 10 minutes. Permeabilize the sections with 200 microliters of PBST for 10 minutes at room temperature. Then denature them with 200 microliters of freshly made carefully titrated 2 Normal hydrochloric acid in 1xPBS for 45 minutes at 37 degrees Celsius to optimize 5mC signal.

After denaturation, add 100 microliters of 0.1 Tris-HCl pH 8.3 on the retinal sections, and incubate for 10 minutes at room temperature to neutralize them. Then, add 500 microliters of blocking solution. And incubate the sections in a humidity chamber at room temperature for one hour.

Add the appropriate antibodies, diluted one to 500 in blocking solution, to the sections and incubate overnight at four degrees Celsius. On the following day, wash the sections three times for 10 to 15 minutes each with 0.1%PBST. Add appropriate secondary antibodies diluted one to 1000 in blocking solution containing DAPI solution at room temperature for one hour.

Next, wash the slides three times with one milliliter of 0.1%PBST for 10 minutes each. After the last wash, add aqueous mounting medium. Cover with a coverslip.

And then proceed with confocal microscopy. After retinal immunostaining at E16, 5mC signal was strong in chromocenters of the cell nuclei and present at a much lower level in the whole cell nuclei. 5hmC staining was exclusively found in the cell nuclei and absent from the chromocenters.

In P0 retina, both 5mC and 5hmC signals were present in the outer neuroblast layer, and inner neuroblast layer. 5mC signal was strong in the chromocenters and nuclear periphery of the cell nuclei, and weaker in between the chromocenters of cell nuclei. 5hmC staining was restricted to the cell nucleus.

In P15 retina, in the outer nuclear layer, 5mC signal was detected in the chromocenters of the cell nuclei and nuclear periphery. Whereas 5hmC signal was in the whole cell nuclei except for the chromocenters. In the inner nuclear, and ganglion cell layer, 5mC signal was strong in the chromocenters of the cell nuclei, and weak in whole cell nuclei.

Whereas the 5hmC signal was found in the whole cell nuclei except for the chromocenters. In adult rod photoreceptors, the 5mC signal was limited to the chromocenter and nuclear periphery, whereas 5hmC signal was restricted to the nuclear periphery. While attempting this procedure, it's important to remember to treat histological sections with the optimal exposure of HCl, which is 30 minutes.

If the exposure is less or more, then optimal results can not be reproducible. Following this procedure, other methods, like high pressure liquid chromatography, mass spectrometry can be performed in order to answer additional questions like quantification of changes in measuring 5-methylcytosine and 5-hydroxymethylcytosine. After its development, this technique paved the way for researchers in the field of neural development to explore epigenetic changes in chromatin.

Don't forget that working with HCl and PFA can be extremely hazardous, and precautions, such as proper disposal, should always be taken while performing this procedure.

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