Immunofluorescence Staining in Mouse Brain Tissue Sections

Staining of 35μm free-floating mouse brain sections is performed in glass staining dishes (Pyrex 36754-60) using 8-section staining nets (Ted Pella 36154-64). dx.doi.org/10.17504/protocols.io.5jyl8pzk9g2w/v1

The sections can be transferred between wells using a paint brush.

Volume of solution:

• 20mL-30mL for antibodies.
• 50mL for washing.

Wash sections 3 times (5 min each wash) in Phosphate Buffer Saline (PBS) at Room temperature to remove cryo-protectant solution.

Wash sections for 0h 5m 0s in Phosphate Buffer Saline (PBS) at Room temperature to remove cryo-protectant solution (1/3).

Wash sections for 0h 5m 0s in Phosphate Buffer Saline (PBS) at Room temperature to remove cryo-protectant solution (2/3).

Wash sections for 0h 5m 0s in Phosphate Buffer Saline (PBS) at Room temperature to remove cryo-protectant solution (3/3).

Wash sections 3 times (5 min each wash) in PBS + 0.1% triton X-100.

Wash sections for 0h 5m 0s in PBS + 0.1% triton X-100 (1/3).

Wash sections for 0h 5m 0s in PBS + 0.1% triton X-100 (2/3).

Wash sections for 0h 5m 0s in PBS + 0.1% triton X-100 (3/3).

Block sections for 1h 0m 0s in PBS + 10% Normal Goat Serum (NGS) + 0.4% Bovine Serum Albumin (BSA) and 0.2% triton X-100 for 1h 0m 0s at Room temperature.

Incubate sections with primary antibodies in blocking solution (PBS + 0.1% triton X-100, 0.4% BSA) 1h 0m 0s at 4°C.

Wash sections 3 times (10 min each wash) in PBS + 0.1% triton X-100 at Room temperature.

Wash sections for 0h 10m 0s in PBS + 0.1% triton X-100 at Room temperature (1/3).

Wash sections for 0h 10m 0s in PBS + 0.1% triton X-100 at Room temperature (2/3).

Wash sections for 0h 10m 0s in PBS + 0.1% triton X-100 at Room temperature (3/3).

Incubate with fluorescent secondary antibodies (1:500) diluted in PBS + 0.1% triton X-100 + 0.4% BSA for 2 hours at Room temperature or 24h 0m 0s at 4°C.

Wash sections 3 times (10 min each wash) in PBS + 0.1% triton X-100 at Room temperature.

Wash sections for 0h 10m 0s in PBS + 0.1% triton X-100 at Room temperature (1/3).

Wash sections for 0h 10m 0s in PBS + 0.1% triton X-100 at Room temperature (2/3).

Wash sections for 0h 10m 0s in PBS + 0.1% triton X-100 at Room temperature (3/3).

Incubate with DAPI in PBS (1:5000) for 0h 30m 0s at Room temperature.

Wash sections 3 times (10 min each wash) in PBS at Room temperature.

Wash sections for 0h 10m 0s in PBS at Room temperature (1/3).

Wash sections for 0h 10m 0s in PBS at Room temperature (2/3).

Wash sections for 0h 10m 0s in PBS at Room temperature (3/3).

Mount sections:

Use a petri dish filled with PBS to mount the sections on superfrost plus slides (Fisher Scientific 12-550-15).

This is easiest to do using a medium sized paint brush.

Let sections dry 0h 2m 0s-0h 5m 0s.

Dry slides for 24h 0m 0s at Room temperature.

1. Protect slides from light at this step.
2. Store slides at 4°C.