Hello Marcio santana Aquino

You stain the cells with the anti-CD20 antibody before fixation. The harsh conditions of fixation can alter or mask the CD20 epitope, making it difficult for the antibody to bind effectively after fixation.

You may use the protocol (in brief) provided below.

1. Harvest B lymphocyte-containing sample and wash the cells with PBS.

2. Resuspend the cell pellet in buffer containing your fluorescently labeled anti-CD20 antibody. Incubate for 20–30 minutes on ice or at 4°C, protected from light.

3. Wash the cells with buffer to remove unbound antibodies.

4. Resuspend the cells in 1–4% PFA and incubate for 15–30 minutes at 4°C.

5. Wash the cells with PBS. At this point, the cells are stable and can be stored at 4°C for up to 24 hours.

6. Resuspend the fixed cells in a permeabilization buffer containing the DNA-labeling dye (like PI or DAPI) and incubate for 15–30 minutes at room temperature, protected from light.

7. Analyze the cells using flow cytometer. The anti-CD20 fluorescence will identify the B lymphocytes, and the DNA dye fluorescence will provide the DNA index for cell cycle analysis within this specific population.

Best,