TITLE: Immunofluorescence Staining in Mouse Brain Tissue Sections
AUTHORS: madalynn.erb Erb

[DESCRIPTION]
This protocol details the immunofluorescence staining in mouse brain tissue sections.

[STEPS]
SECTION: Day 1
1. 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
SECTION: Day 1
1.1. The sections can be transferred between wells using a paint brush. 
SECTION: Day 1
1.2. Volume of solution: 

20 mL-30 mL for antibodies.
50 mL for washing.
SECTION: Day 1
2. Wash sections 3 times (5 min each wash) in Phosphate Buffer Saline (PBS) at Room temperature to remove cryo-protectant solution.
SECTION: Day 1
2.1. Wash sections for 5 min in Phosphate Buffer Saline (PBS) at Room temperature to remove cryo-protectant solution (1/3).
SECTION: Day 1
2.2. Wash sections for 5 min in Phosphate Buffer Saline (PBS) at Room temperature to remove cryo-protectant solution (2/3).
SECTION: Day 1
2.3. Wash sections for 5 min in Phosphate Buffer Saline (PBS) at Room temperature to remove cryo-protectant solution (3/3).
SECTION: Day 1
3. Wash sections 3 times (5 min each wash) in PBS + 0.1% triton X-100.
SECTION: Day 1
3.1. Wash sections for 5 min in PBS + 0.1% triton X-100 (1/3).
SECTION: Day 1
3.2. Wash sections for 5 min in PBS + 0.1% triton X-100 (2/3).
SECTION: Day 1
3.3. Wash sections for 5 min in PBS + 0.1% triton X-100 (3/3).
SECTION: Day 2
6. Wash sections 3 times (10 min each wash) in PBS + 0.1% triton X-100 at Room temperature.

SECTION: Day 2
6.1. Wash sections for 10 min in PBS + 0.1% triton X-100 at Room temperature (1/3).
SECTION: Day 2
6.2. Wash sections for 10 min in PBS + 0.1% triton X-100 at Room temperature (2/3).
SECTION: Day 2
6.3. Wash sections for 10 min in PBS + 0.1% triton X-100 at Room temperature (3/3).
SECTION: Day 2
7. 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 1440 min at 4 °C.

 

SECTION: Day 2
8. Wash sections 3 times (10 min each wash) in PBS + 0.1% triton X-100 at Room temperature.
SECTION: Day 2
8.1. Wash sections for 10 min in PBS + 0.1% triton X-100 at Room temperature (1/3).
SECTION: Day 2
8.2. Wash sections for 10 min in PBS + 0.1% triton X-100 at Room temperature (2/3).
SECTION: Day 2
8.3. Wash sections for 10 min in PBS + 0.1% triton X-100 at Room temperature (3/3).
SECTION: Day 2
9. Incubate with DAPI in PBS (1:5000) for 30 min at Room temperature.
SECTION: Day 2
10. Wash sections 3 times (10 min each wash) in PBS at Room temperature.
SECTION: Day 2
10.1. Wash sections for 10 min in PBS at Room temperature (1/3).
SECTION: Day 2
10.2. Wash sections for 10 min in PBS at Room temperature (2/3).
SECTION: Day 2
10.3. Wash sections for 10 min in PBS at Room temperature (3/3).
SECTION: Day 2
11. Mount sections:	
SECTION: Day 2
11.1. Use a petri dish filled with PBS to mount the sections on superfrost plus slides (Fisher Scientific 12-550-15).
SECTION: Day 2
11.2. This is easiest to do using a medium sized paint brush.
SECTION: Day 2
11.3. Let sections dry 2 min-5 min.

 

SECTION: Day 2
12. Dry slides for 1440 min at Room temperature.

Protect slides from light at this step.
Store slides at 4 °C.
SECTION: Day 1
4. Block sections for 60 min in PBS + 10% Normal Goat Serum (NGS) + 0.4% Bovine Serum Albumin (BSA) and 0.2% triton X-100 for 60 min at Room temperature.

SECTION: Day 1
5. Incubate sections with primary antibodies in blocking solution (PBS + 0.1% triton X-100, 0.4% BSA) 60 min at 4 °C.