I want to analyze the localization of my protein at different phases of the cell cycle. Initially I performed IF staining (with U-87 cell line) and used confocal microscope to detect my protein and get some good results, and then I want to confirm these results by using different cell lines ( SH-SY5Y, C6, A172 and etc.).Unfurtunately, I could not get any results since then because when I perform IF staing and use confocal microscope, I could not detect any mitotic cells, there were only non-dividing cells ( I tried different cell lines) I probably I lose my mitotic cells during the staining process and tried a lot of new ways to solve this problem but I failed again again. I don't understand what the problem is, please help me. My current protocol is below:
1. Grow cells on poly l lysine coated coverslips ( coated with poly-l-lysine and washed with PBS and incubated overnight at RT)
2. Wash with PBS gently and incubate 4 % PFA for 20 minutes, followed by 2 washes with PBS
3.Permeabilise 1o minutes in 0.2 % Triton X-100
4.Block in 3 % BSA OR 10 % FBS in PBS with 0.2 % Triton X-100
5. Incubate with primary antibodies in blocking solution for overnight
6.Wash 3 times with PBS-Triton X-100 and incubate with secondary antibodies for one hour.
7. Wash 3 times with PBS-Triton X-100 again and mount.