Here, we present a protocol to detect 5-hydroxymethylcytosin in cells and the brain tissues using immunofluorescence, the new method, and the DNA dot-blot. 5-hydroxymethylcytosin, i.e. 5hmC, mitigated epigenetic modification, plays important function in regulating gene expression and involved in multiple neurological disorders.
Actually, our methods can be used to detect 5hmC in multiple cells and the tissues. It's convenient and can be performed with common equipments in the lab. Begin by positioning the mouse face up on a plastic board and fixing each limb with sticky tape.
Use surgical scissors to cut through the skin and muscle, and open the thoracic cavity. Expose the heart and cut off a small part of the right atrium with fine surgical scissors. Using a 10-milliliter disposable, sterilized syringe, perfuse the mouse from the left ventricle with cold PBS.
Then, perfuse the mouse with 4%PFA until it is stiff. Then, open the skull with bone forceps and remove the brain. Place the brain into a 15-milliliter centrifuge tube filled with five milliliters of 4%PFA and store it at four degrees Celsius.
After 24 hours, transfer the brain into a 30%sucrose solution and leave it at four degrees Celsius for complete dehydration. Prior to sectioning, imbed the brain in optimal cutting temperature compound and cool to minus 20 degrees Celsius for at least one hour. Section brain samples at a thickness of 20 to 40 micrometers using a cryostat microtome.
Collect sections into PBS and store at four degrees Celsius. Pick up the brain sections of interest and put them into a 24-well plate with PBS. Wash sections with PBS on a shaker for 10 minutes.
Remove the PBS and treat with preheated one-molar hydrochloric acid at 37 degrees Celsius for 30 minutes. Hydrochloric acid is corrosive and please prepare it in the chemical hold. Then, wash the samples three times with PBS for five minutes and block them with PBS containing 3%normal goat serum and 0.1%Triton X-100.
Leave the samples on a shaker at room temperature for one hour. Add specific primary antibodies to the sections and incubate overnight at four degrees Celsius on a shaker. The next day, take the samples out and leave them at room temperature for one hour.
Wash the samples three times with PBS and add secondary antibodies. Cover the plate with aluminum and leave it at room temperature for one hour while shaking. After the incubation, wash the samples three times with PBS and mount them onto slides.
Add 100 to 150 microliters of anti-fade mounting medium, cover with a premium cover glass, and seal with nail polish. Image the brain sections with a regular or confocal microscope. To isolate genomic DNA, start by dissecting the hippocampus, cortex, and cerebellum tissues on an ice-cooled dish.
Add one milliliter of DNA lysis buffer and grind the tissues. Transfer the sample into a clean microcentrifuge tube and add 250 micrograms of proteinase-K for every 600 microliters of lysis buffer. Then, add about 50 micrograms of RNase A per sample and incubate for at least 12 hours at 37 degrees Celsius.
After the incubation, add an equal volume of phenol chloroform isoamyl alcohol and mix thoroughly. Centrifuge the mixture according to manuscript directions and transfer the supernatant to a new tube. Add 600 microliters of chloroform to the supernatant, mix thoroughly, and centrifuge according to manuscript directions.
Transfer the supernatant to a new tube and add 500 microliters of isopropanol. Mix thoroughly and centrifuge again to precipitate the DNA. After the centrifugation, discard the supernatant, and wash the DNA pellet with 70%ethanol according to manuscript directions.
Air dry the DNA pellet completely and then, dissolve it in Tris-HCl buffer. Prior to starting, prepare the required solutions and sample mixture according to manuscript directions. Denature the DNA sample by heating it to 100 degrees Celsius for 10 minutes and then, place it on ice.
Cut the appropriate size of a nylon membrane and rinse it with 6X SSC. Place the membrane on a dot-blot apparatus and connect the vacuum pump. Spot six-microliter dots onto the membrane and hybridize for 30 minutes at 80 degrees Celsius.
Then, block the sample membrane with fat-free milk and Tris-buffered saline, or TBS, for one hour. Add the polyclonal rabbit anti-5-hydroxymethylcytosin antibody to the membrane and incubate overnight at four degrees Celsius. The next day, leave the sample membrane at room temperature for one hour, then wash the membrane three times with TBS.
Incubate the membrane with the anti-rabbit secondary antibody for 30 minutes and then, wash three times with TBS. Visualize the chemiluminescence signals and quantify their intensity. This protocol can be used to visualize 5-hydroxymethylcytosin, or 5hmC, in the hippocampus of adult mice.
Immunofluorescence with antibodies against neuronal cells, the 5hmC reveals that there is an enrichment of 5hmC in neurons. Dynamics of 5hmC during neuronal development can be determined with a dot-blot assay of DNA samples isolated from proliferating and differentiated adult neural stem cells. Global levels of 5hmC significantly increase during differentiation and the level of 5hmC in neurons is higher than in neural stem cells.
The key step of this method is to make sure that complete denaturation of DNA samples.
