Immunofluorescence staining protocol with Antigen Retrieval

Staining protocol for 35 µm free floating mouse brain sections.

Wash tissue sections 3 times (5 minutes each wash) in PBS to remove cryoprotectant solution.

Wash tissue sections for 0h 5m 0s in PBS to remove cryoprotectant solution (1/3).

Wash tissue sections for 0h 5m 0s in PBS to remove cryoprotectant solution (2/3).

Wash tissue sections for 0h 5m 0s in PBS to remove cryoprotectant solution (3/3).

Mount tissue onto positively charged slides.

Allow slides to air dry then place in oven set at 37°C-50°C 0h 5m 0s.

If you would like to use a PAP pen to draw a box around your tissue sections, do that now and wait for slides to dry.

Wash slides in PBS for 0h 5m 0s.

Preheat steamer with a Coplin jar containing the antigen retrieval buffer until temperature reaches 95°C-100°C (about 0h 10m 0s

Immerse slides into the preheated buffer in the steamer for 0h 30m 0s. 

After 30 minutes turn off the steamer but allow to cool to Room temperature (about 20-0h 30m 0s) before progressing to the next step. 

Wash slides in PBS 0.1% Trition X-100 (PBST) 2 x 2 min. 

Wash slides in PBS 0.1% Trition X-100 (PBST) for 0h 2m 0s (1/2).

Wash slides in PBS 0.1% Trition X-100 (PBST) for 0h 2m 0s (2/2).

Wash slides in PBS 3 x 5 min.

Wash slides in PBS for 0h 5m 0s (1/3).

Wash slides in PBS for 0h 5m 0s (2/3).

Wash slides in PBS for 0h 5m 0s (3/3).

Block for 2h 0m 0s at Room temperature in PBS (0.4% BSA, 10% Goat normal serum, 0.3% triton X-100).

Incubate 0h 10m 0s with primary antibody in PBS (2% Goat normal serum, 0.3% triton X-100) 0h 10m 0s at Room temperature. 

Wash slides in PBST 3 x 10 min.

Wash slides in PBST for 0h 10m 0s (1/3).

Wash slides in PBST for 0h 10m 0s (2/3).

Wash slides in PBST for 0h 10m 0s (3/3).

Incubate slides with secondary antibody in PBS for 4h 0m 0s at Room temperature or at 4°C 4h 0m 0s. 

Wash slides in PBST 3 x 10 min. 

Wash slides in PBST for 0h 10m 0s (1/3).

Wash slides in PBST for 0h 10m 0s (2/3).

Wash slides in PBST for 0h 10m 0s (3/3).

Incubate slides with Hoescht stain (1:5000) for 0h 30m 0s at Room temperature.

Wash slides in PBST 3 x 10 min.

Wash slides in PBST for 0h 10m 0s (1/3).

Wash slides in PBST for 0h 10m 0s (2/3).

Wash slides in PBST for 0h 10m 0s (3/3).

Mount slides using ProLong™ Diamond Antifade Mountant.

Dry slides on a flat surface at Room temperature in the dark.

Store slides at 4°C the next day.