Immunofluorescence assay for 5hmc

Plate cells in 24-well plates with coverslips at 10,000–50,000 cells per well. After desired treatments, the next steps should be follow:

1)    Fix with 4% paraformaldehyde for 30 min to 60 min. depending of cell type. (Cells that do not attach well to glass are leave longer).

2)    Incubate with 2N HCl at 37°C for 20 min. The 2N HCL is prewarmed to 37°C. Enough to cover slides. The timing is very important and there is some variation depending of the cell type but most of them give a good staining within 18 to 20 min. 

3)    Neutralize with 100 μM Tris-HCl pH 8.5 for 10 min. Abundant to make sure it cover any HCL left on the wall.  

4)    Wash with PBS 3 times. 5 minutes incubation at room temperature between washes Use abundant PBS.

5)     Block with blocking solution: 0.4% Triton X-100, 10% BSA in PBS for 1 hr. Nuclear membrane permeabilization is very important for the antibody to reach the nuclei.

6)     Incubate cells in blocking solution with anti-5-hmC antibody (Active Motif #39769, 1:1,000) at 4°C overnight. Recently we are using 1:500 of the same Active Motif antibody due to a variation with previous lots.

7)    Wash with PBS 4 times. 5 minutes incubation at room temperature between washes Use abundant PBS.

8)    Incubate with Alexa Fluor 488-conjugated donkey anti-rabbit IgG (1:500) and DAPI (1ug/ml) diluted in blocking buffer at room temperature for 1 hour. (Note: longer HCL incubation in step 2 will damage the DAPI staining. Shorter HCL incubation will result in not good 5hmC staining).

9)    Wash with PBS 4 times. 5 minutes incubation at room temperature between washes. Use abundant PBS.

10)  Mount with Mowiol 4-88 mounting medium or any suitable mounting media. Take Images using confocal focusing in the 5hmC. 

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