Immunofluorescence protocol for FFPE human post-mortem brain sections to detect alpha-synuclein and tau pathology

Place slides with FFPE human brain tissues on a slide rack and bake in a 60°C oven for 1h 0m 0s

2. Continue using the slide rack, submerge slides in the following solution to de-paraffinize:

   a. HistoChoice: 2 x 0h 7m 0s

   b. 100% ethanol: 2 x 0h 3m 0s

   c. 95% ethanol: 0h 3m 0s

   d. 70 % ethanol: 0h 3m 0s

3. Submerge in MiliQ/ultrapure water for 0h 3m 0s.

4. Tap gently sideways on flat surface covered with paper towels to remove excess water.

5. Submerge slides in 70% Formic Acid for 0h 20m 0s.

6. Wash in MiliQ/ultrapure water for 2x0h 5m 0s.

7. Remove slides from the rack and submerge slides directly into 0.01Molarity (M) sodium citrate buffer (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

of2h 0m 0s .

9. Wash with MiliQ/ultrapure water for 0h 1m 0s, followed by washing with 1 X PBS for 0h 5m 0s.

10. Prepare 0.1% Sodium borohydride (NaBH₄) in 1xPBS for 0h 30m 0s of quenching. The solution must always remain chilled in ice.

11. Wash in 1x PBS for 2x0h 5m 0s

12. Add Human FC blocker in 1X PBS with a ratio of 1:50, incubate slides with Human FC blocker at Room temperature for 0h 5m 0s 

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 TrueBlac^®‱Background Suppressor to completely cover sample. Room temperature``0h 30m 0s

14. Remove the Background Suppressor and add IF Blocking Buffer (home-made).Room temperature 1h 0m 0s

5. Prepare 150 ul per sample of primary antibody solution consisting of selected primary antibody diluted in home-made blocking buffer.

A	B
SNCA (Gt)	1:200
Ubq (Rb)	1:100
P62 (Ms IgG1)	1:100

Primary antibody table and dilutions

16. Prepare humidified chamber and remove Blocking buffer by tapping on paper towel.

17. Add primary antibody diluted in Blocking buffer and incubate for 48h 0m 0s in 4°C

1. Wash slides in PBST 3x0h 10m 0s. 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.

A	B
Dn anti Gt-AF800	1:250
Dn anti Rb-AF647	1:200
Dn anti Ms-AF488	1:200
Hoescht-405	1:500

Midbrain and cortical secondary antibodies and dilutions

3. Incubate sample in secondary antibody in Room temperature for 2h 0m 0s

4. Wash slides in PBST 3x0h 5m 0s, followed by 1x PBS 1x0h 5m 0s

5. Prepare conjugated antibodies using Zenon^TM labeling kits within0h 30m 0s before applying the antibodies.

A	B
AT8(Ms, IgG1)	Zenon‱ Ms IgG1 Labeling Kits (AF594)
TH (Rb)	Zenon‱ Rb Labeling Kits (AF532)

Midbrain conjugated antibodies

A	B
AT8(Ms, IgG1)	Zenon‱ Ms IgG1 Labeling Kits (AF594)
HuD (Rb)	Zenon‱ Rb Labeling Kits (AF532)

Cortical conjugated antibodies

6. Incubate sample in conjugated antibodies in Room temperature for 2h 0m 0s

7. Wash slides in 1x PBS 3x0h 5m 0s

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 3x0h 5m 0s

12. Remove as much moisture without drying tissue using Kimwipes

13. Retrieve Mounting with EverBrite^TM 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^TM Coverslip Sealant.

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