TITLE: Immunofluorescence staining protocol with Antigen Retrieval
AUTHORS: madalynn.erb Erb

[DESCRIPTION]
This protocol details the immunofluorescence staining protocol with antigen retrieval.

[STEPS]
SECTION: Day 1
1. Staining protocol for   free floating mouse brain sections.

SECTION: Day 1
2. Wash tissue sections 3 times (5 minutes each wash) in PBS to remove cryoprotectant solution.

SECTION: Day 1
3. Mount tissue onto positively charged slides.
SECTION: Day 1
4. Allow slides to air dry then place in oven set at 37 °C-50 °C 5 min.

 

SECTION: Day 2
5. 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.
SECTION: Day 2
6. Wash slides in PBS for 5 min.

SECTION: Day 2
7. Preheat steamer with a Coplin jar containing the antigen retrieval buffer until temperature reaches 95 °C-100 °C (about 10 min

 

SECTION: Day 2
8. Immerse slides into the preheated buffer in the steamer for 30 min. 

SECTION: Day 2
9. After 30 minutes turn off the steamer but allow to cool to Room temperature (about 20-30 min) before progressing to the next step. 

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

SECTION: Day 2
11. Wash slides in PBS 3 x 5 min.

SECTION: Day 2
12. Block for 120 min at Room temperature in PBS (0.4% BSA, 10% Goat normal serum, 0.3% triton X-100).

 

SECTION: Day 2
13. Incubate 10 min with primary antibody in PBS (2% Goat normal serum, 0.3% triton X-100) 10 min at Room temperature. 

SECTION: Day 3
14. Wash slides in PBST 3 x 10 min.

SECTION: Day 3
15. Incubate slides with secondary antibody in PBS for 240 min at Room temperature or at 4 °C 240 min. 

SECTION: Day 3
16. Wash slides in PBST 3 x 10 min. 

SECTION: Day 3
17. Incubate slides with Hoescht stain (1:5000) for 30 min at Room temperature.

SECTION: Day 3
18. Wash slides in PBST 3 x 10 min.

SECTION: Day 3
19. Mount slides using ProLong™ Diamond Antifade Mountant.
SECTION: Day 3
20. Dry slides on a flat surface at Room temperature in the dark.

 

SECTION: Day 3
21. Store slides at 4 °C the next day.

SECTION: Day 1
2.1. Wash tissue sections for 5 min in PBS to remove cryoprotectant solution (1/3).
SECTION: Day 1
2.3. Wash tissue sections for 5 min in PBS to remove cryoprotectant solution (3/3).
SECTION: Day 1
2.2. Wash tissue sections for 5 min in PBS to remove cryoprotectant solution (2/3).
SECTION: Day 2
10.1. Wash slides in PBS 0.1% Trition X-100 (PBST) for 2 min (1/2).
SECTION: Day 2
10.2. Wash slides in PBS 0.1% Trition X-100 (PBST) for 2 min (2/2).
SECTION: Day 2
11.1. Wash slides in PBS for 5 min (1/3).

SECTION: Day 2
11.2. Wash slides in PBS for 5 min (2/3).
SECTION: Day 2
11.3. Wash slides in PBS for 5 min (3/3).
SECTION: Day 3
14.1. Wash slides in PBST for 10 min (1/3).

SECTION: Day 3
14.2. Wash slides in PBST for  10 min (2/3).
SECTION: Day 3
14.3. Wash slides in PBST for  10 min (3/3).
SECTION: Day 3
16.1. Wash slides in PBST for 10 min (1/3).

SECTION: Day 3
16.2. Wash slides in PBST for 10 min (2/3).
SECTION: Day 3
16.3. Wash slides in PBST for 10 min (3/3).
SECTION: Day 3
18.1. Wash slides in PBST for 10 min (1/3).

SECTION: Day 3
18.2. Wash slides in PBST for 10 min (2/3).
SECTION: Day 3
18.3. Wash slides in PBST for 10 min (3/3).