TITLE: Immunofluorescence protocol for FFPE human post-mortem brain sections to detect alpha-synuclein and tau pathology
AUTHORS: Felicia Suteja, Hongyun Li, YuHong Fu

[DESCRIPTION]
This protocol details the method for preparing and staining human formalin-fixed, paraffin embedded post mortem brain tissue to detect alpha-synuclein and tau pathology using immunofluorescence.

[BEFORE_START]
Formalin-Fixed Paraffin-Embedded (FFPE) sections of the human midbrain and cortical regions were cut at 6um with a Leica HM325 rotary microtome and mounted on Series 2 adhesive microscope slides.

[STEPS]
SECTION: Day I - Tissue Prep
1. Place slides with FFPE human brain tissues on a slide rack and bake in a 60 °C oven for 60 min

SECTION: De-paraffinize
2. 2. Continue using the slide rack, submerge slides in the following solution to de-paraffinize: 
      a. HistoChoice: 2 x 7 min
      b. 100% ethanol: 2 x 3 min 
      c. 95% ethanol: 3 min 
      d. 70 % ethanol: 3 min

3. Submerge in MiliQ/ultrapure water for 3 min.

4. Tap gently sideways on flat surface covered with paper towels to remove excess water. 
SECTION: Antigen retrieval
3. 5. Submerge slides in 70% Formic Acid for 20 min.

6. Wash in MiliQ/ultrapure water for 2x5 min.

7. Remove slides from the rack and submerge slides directly into 0.01 Molarity (M) sodium citrate buffer (pH 6) and incubate sections in a programmable antigen retrieval cooker

8. Let the pressure cooker reach its peak of 121 °C before gradually cooling for a total
of 120 min. 

9. Wash with MiliQ/ultrapure water for 1 min, followed by washing with 1 X PBS for 5 min.

SECTION: Quenching aldehyde group
4. 10. Prepare 0.1% Sodium borohydride (NaBH4) in 1xPBS for 30 min of quenching. The solution must always remain chilled in ice. 

11. Wash in 1x PBS for 2x5 min

SECTION: Human Fc blocker treatment
5. 12. Add Human FC blocker in 1X PBS with a ratio of 1:50, incubate slides with Human FC blocker at Room temperature for 5 min 



SECTION: Background Suppressor system treatment
6. Component A (Background Suppressor) may become turbid or form a gel at 4 °C this does not affect performance. Warm the buffer to room temperature or 37 °C until clear (light blue) and completely
liquid before use.
     
13. Add enough TrueBlack‱Background Suppressor to completely cover sample.  Room temperature30 min
      
14. Remove the Background Suppressor and add IF Blocking Buffer (home-made). Room temperature 60 min

SECTION: Primary antibody incubation
7. 5. Prepare 150 ul per sample of primary antibody solution consisting of selected primary antibody diluted in home-made blocking buffer. 
 
 
 
16. Prepare humidified chamber and remove Blocking buffer by tapping on paper towel.

17. Add primary antibody diluted in Blocking buffer and incubate for 2880 min in 4 °C

SECTION: Day 4 - Secondary antibodies
8. 1. Wash slides in PBST 3x10 min. Slides must be protected from light from this step. 

2. Prepare 150 ul per sample of secondary antibody solution consisting of selected secondary antibody diluted appropriately in home-made blocking buffer.
 
 
3. Incubate sample in secondary antibody in Room temperature for 120 min

4. Wash slides in PBST 3x5 min, followed by 1x PBS 1x5 min

SECTION: Conjugated antibodies
9. 5. Prepare conjugated antibodies using Zenon‱ labeling kits within30 min before applying the antibodies.
 
 
 
 
6. Incubate sample in conjugated antibodies in Room temperature for 120 min

7. Wash slides in 1x PBS 3x5 min

SECTION: Post-treatment with TrueBlack® Lipofuscin Autofluorescence Quencher
10. 8. Prepare 150 ul per sample of 1xTrueBlack‱ Lipofuscin Autofluorescence Quencher in 70% ethanol.

9. Tap slides to paper towels to remove excess washing solution and then place in humidified chamber. 

10. Add diluted TrueBlack‱ solution and incubate for 30-60 seconds. 

11. Rinse slides in 1xPBS 3x5 min

SECTION: Mounting and cover slipping
11. 12. Remove as much moisture without drying tissue using Kimwipes

13. Retrieve Mounting with EverBrite‱ Mounting Medium to warm at Room temperature before dispensing approximately 10 - 20 ml on slides.

14. Place coverslip gently on the slides and wait for the Mounting Medium to dry before applying the perimeter of the coverslip with Biotium’s CoverGrip‱ Coverslip Sealant.

15. Store in a protected slide boxaway from light at 4 °C