I did a frozen section of mouse brain tissue with immunofluorescence staining. My partner and I did c-fos staining together. We used different primary and secondary antibodies for different research species, but the subsequent staining steps were the same. However, there were a lot of noise and asymmetry in my tissues, and the results of my partner were very good.

1, hydration: room temperature recovery 10min, PBS rinse, 5 min × 3;

2, permeability: brain tablet drops 0.2% Trition X-100, 10min; Rinse with PBS, 5 min × 3;

3. Block: 10% of normal serum from host source of secondary antibody +0.3M (22.52 mg/mL) glycine, block at room temperature for 1-2h.

4, at room temperature, primary antibody, 4 ℃ overnight incubation.

5, PBS washing, 5 min × 5

6, at room temperature, incubation with two antibodies to avoid light 4 spend the night. Rinse with PBS, 5 min ×6