The overall goal of this immunochemical protocol is to evaluate the spatial distribution of modified forms of cytosine in various tissue and cellular context based on the use of peroxidase conjugated secondary antibodies and tyramide signal amplification. This method overcomes the limitation of other techniques that do not provide spatial information necessary for understanding the biological function of modified forms of cytosine. In addition, this method permits co-detection of modified forms of cytosine with protein lineage markers and can be employed to study their nuclear localization.
To begin this procedure, fix rehydrated tissue sections of wild-type CD1 mouse embryos and adult brain tissues by placing them in either ice cold 4%PFA or 4%FA for 15 minutes at room temperature. Then, remove the excess fixative by washing the sections in PBS for five minutes at room temperature. Next, permeabilize the tissue sections by placing them in a Coplin jar filled with PBX for 30 minutes at room temperature.
Subsequently, remove the excess PBX by washing the sections shortly in PBT. Now, place the permeabilized sections in 2 N HCl for 60 minutes at room temperature for DNA depurination. Then, transfer the sections to 10 millimolar Tris-Hcl for 30 minutes at room temperature to neutralize the HCl.
Alternatively, wash the sections three times for five minutes each in PBS. Incubate the sections in PBT for five minutes at room temperature. After that, carefully remove the liquid from the area surrounding the tissue sections.
In the meantime, keep the tissue sections moist. Use a hydrophobic barrier pen to encircle the sections without touching them. Subsequently, incubate the sections in 100 microliters of blocking solution for one hour at room temperature in a humid chamber.
Then, incubate the tissue sections in 100 microliters of a 1:5000 dilution of a mouse monoclonal anti-5hmC primary antibody and 1:1000 dilution of a rabbit polyclonal anti-5caC primary antibody in blocking solution for one hour at room temperature. Alternatively, perform the incubation overnight at four degrees Celsius. Next, remove excess antibodies by washing the sections in a Coplin jar filled with PBT three times for five minutes each at room temperature.
Then, remove excess PBT, and if necessary, encircle the sections again with a hydrophobic barrier pen as PBT contains detergent that can weaken the hydrophobic barrier. Following that, make a 1:400 dilution of goat anti-rabbit HRP-conjugated antibody and a 1:400 dilution of donkey anti-mouse 555-conjugated antibody in blocking solution. Then incubate the tissue sections in 100 microliters of the secondary antibody mixture for one hour at room temperature in a humid chamber.
Afterward, wash the tissue sections in a Coplin jar filled with PBT three times for five minutes each at room temperature. Then transfer the tissue sections to 100 microliters of tyramide at a 1:200 dilution in the tyramide signal amplification buffer for two minutes at room temperature. The incubation time was the tyramide solution should be optimized experimentally for each individual batch of tyramide signal amplification kit where a linear relationship between signal intensity and the duration of tyramide-based signal amplification is observed.
Immediately after, remove excess tyramide solution by washing the slides three times for five minutes each in PBT. Carefully remove excess PBT and immediately cover the sections with a drop of mounting medium. Gently place a cover slip on the tissue sections and seal the cover slip with nail polish.
Then keep the tissue sections at four degrees Celsius for several hours before microscopic examination. To determine the distribution of 5hmC in brain tissue sections, the co-detection of this epigenetic modification was performed with a marker for post-mitotic neurons, NeuN. Immunohistochemical analysis revealed that whereas the prominent 5hmC staining colocalized with NeuN-positive cells, NeuN-negative glial cells possessed lower levels of genomic 5hmC.
To determine the distribution of 5caC in differentiating neural stem cells, costaining of this marker was performed with a glial marker, GFAP, on the fixed cultures of neural stem cells three days after the induction of glial differentiation. Unlike in neural stem cells or mature astrocytes, strong 5caC signal in a large proportion of cells expressing GFAP was observed. Once mastered, this technique can be done in about eight hours if it is performed properly.
While attempting this procedure, it is important to remember to have all reagents and materials together with your samples ready. Following this procedure, confocal imaging can be performed in order to answer additional questions about nuclear distribution of modified forms of cytosine. This can contribute to deciphering their potential biological function.
Don't forget that working with 2 N Hcl can be extremely hazardous and precautions such as safety cabinet and protective wear should always be taken while performing this procedure.
