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

All the animal procedures have been approved by the Animal Ethics Committee of Zhejiang University.

1. The Culture of Adult Neural Stem Cells and Neurons

1. Isolate adult neural stem cells from the forebrain of an adult (8-10 week old) C57/BL6 male mouse as described previously^31,32.
2. Culture adult neural stem cells in DMEM/F-12 medium containing 20 ng/mL FGF-2, 20 ng/mL EGF, 2% B27 supplement, 1% antibiotic-antimycotic, and 2 mM L-Glutamine in a 5% CO₂ incubator at 37 °C. Induce the differentiation of adult neural stem cells with 1 µM retinoic acid and 5 µM forskolin for 48 h as described previously^31,32.
3. Isolate neurons from the embryonic day 17 (E17) hippocampi of the mouse and culture with neurobasal medium containing of 0.25% L-Glutamine, 0.125% GlutaMax and 2% B27 supplement in a 5% CO₂ incubator at 37 °C as previously described³³.

2. Transcardial Perfusion of the Mouse

1. Prepare 10% chloral hydrate, 4% paraformaldehyde (PFA) and phosphate buffered saline (PBS) one day before the experiment, and store at 4 °C.
2. Anesthetize an adult male or female C57 BL/6J mouse with 10% chloral hydrate (50 mg/kg, i.p.), and ensure that the animal is deeply anesthetized by checking the body reaction. Fix each limb with sticky tape on a plastic board (in a face-up position).
3. Cut the skin and then the muscle with surgery scissors. Open the thoracic cavity with a surgical scissor. Expose the heart and cut off a little part of the right atrium with a fine surgical scissor.
4. Perfuse the mouse with a 10 mL disposable sterilized syringe from the left ventricle with cold PBS (around 30 mL per adult mouse).
5. Perfuse the mouse with 4% PFA (around 30 mL in 10 min per adult mice) until it is stiff.
6. Open the skull of the mouse with bone forceps. Remove the brain and put into 5 mL of 4% PFA in a 15 mL centrifuge tube for post-fixation at 4 °C.
   NOTE: Clean the surgical area after surgery has finished.
7. At least 24 h later, transfer the brain samples into a 30% sucrose solution for complete dehydration at 4 °C.

3. Brain Sectioning

1. Embed the brain samples in optimal cutting temperature compound (OCT) in a small container, and cool down at -20 °C for at least 1 h.
2. Section brain samples at a thickness of 20-40 μm with a cryostat microtome.
3. Collect sections into PBS and store at 4 °C.

4. Immunofluorescence Staining

1. Pick up the sections with the targeted brain regions and put them into a 24-well plate with PBS. For cultured cells on a coverslip, go directly to step 4.2.
2. Wash with PBS on the shaker at room temperature for 10 min. Repeat this twice more.
3. Remove PBS, and treat with preheated 1 M HCl for 30 min at 37 °C.
   NOTE: Prepare 1 M HCl by adding 1 mL of hydrochloric acid (36-38%) into 10 mL of water in a chemical hood. Preheat 1 M HCl in the incubator at 37 °C.
4. Wash the samples with PBS for 5 min. Repeat this twice more.
5. Block samples with PBS containing 3% normal goat serum and 0.1% Triton X-100 for 1 h on the shaker at room temperature.
6. Incubate sections with specific primary antibodies overnight at 4 °C on the shaker.
   NOTE: Use the following primary antibodies: polyclonal rabbit antibodies anti-5-hydroxymethylcytosine (1:5,000), mouse monoclonal antibody anti-NeuN (1:500).
7. Take out the samples, and further incubate at room temperature for 1 h.
8. Wash the samples with PBS for 10 min. Repeat this twice more.
9. Incubate with secondary antibodies corresponding to the primary antibodies at room temperature for 1 h on the shaker. Cover the plate with aluminum.
   NOTE: Use the following secondary antibodies: Alexa Fluor 488 goat anti-rabbit IgG (1:500), Alexa Fluor 568 goat anti-mouse IgG (1:500). Counterstain nuclei with 4′-6-diamidino-2-phenylindole (DAPI).
10. Wash the samples with PBS for 10 min on the shaker. Repeat this twice more.
11. Mount brain sections onto the slides, add proper amount of antifade mounting medium (around 100-150 μL), and cover with premium cover glass. Seal with nail polish.
12. Take images with a regular or confocal fluorescence microscope.

5. Genomic DNA Isolation

1. Euthanize an adult C57 BL/6J mouse (male or female) by cervical dislocation and remove the brain.
2. Dissect the hippocampus, cortex and cerebellum tissues on an ice-cooled dish. Grind tissues with a tissue grinder in 1 mL of DNA lysis buffer and transfer into clean microcentrifuge tube. Add 250 μg of proteinase K per 600 μL of lysis buffer. For cell pellets, directly add lysis buffer, proteinase K, and mix thoroughly.
   NOTE: Prepare DNA lysis buffer: 5 mM EDTA, 0.2% SDS, 200 mM NaCl in 100 mM Tris-HCl, pH 8.5. Wash and autoclave the tissue grinder before the experiment.
3. The second day, add about 50 μg of RNase A per sample for at least 12 h at 37 °C.
4. Add an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1) and mix completely.
5. Centrifuge at 20,817 x g for 15 min, and remove the supernatant into a new microcentrifuge tube.
6. Add 600 μL of chloroform to the supernatant to precipitate DNA, and mix thoroughly.
7. Centrifuge at 20,817 x g for 15 min, and remove the supernatant into another new tube.
8. Add 500 μL of isopropanol to the supernatant, and mix thoroughly.
9. Centrifuge at 20,817 x g for 15 min, and remove the supernatant completely.
10. Wash the precipitation with 1 mL of 70% ethanol, centrifuge at 20,817 x g for 1 min, and remove the supernatant completely. Repeat once.
11. Dry the DNA pellet completely.
12. Dissolve DNA pellet with Tris-HCl buffer (pH 8.5) to the proper concentration.

6. DNA Dot Blot

1. Prepare the solutions: 2 M NaOH, Tris-HCl buffer (pH 8.5), 6x saline sodium citrate (SSC).
2. Make the sample mixture as Table 1.
3. Denature DNA samples at 100 °C for 10 min, and cool down on ice.
4. Cut the proper size of nylon membrane (e.g., Hybond-N+) and rinse with 6x SSC.
5. Put the membrane on dot-blot apparatus and connect to the vacuum pump. Spot 6 μL of mixture per dot onto the membrane.
6. Hybridize for 30 min at 80 °C, and block the sample membrane with fat-free milk in Tris-buffered saline (TBS) for 1 h.
7. Incubate with primary antibody at 4 °C overnight.
   NOTE: the following primary antibody: polyclonal rabbit anti-5-hydroxymethylcytosine (1:5,000).
8. On the second day, incubate the sample membranes at room temperature for 1 h. Wash with TBS for 10 min. Repeat this wash twice more.
9. Incubate the membrane with anti-rabbit secondary antibody (1:5,000) for 30 min at room temperature.
10. Wash with TBS for 10 min. Repeat this wash twice more.
11. Visualize the chemiluminescence signals, and quantify signal intensities.