Aug 22, 2024
Version 2
Immunofluorescence protocol for FFPE human post-mortem brain sections to detect alpha-synuclein and tau pathology V.2
- Felicia Suteja1,
- Hongyun Li1,
- YuHong Fu1
- 1University of Sydney
Protocol Citation: Felicia Suteja, Hongyun Li, YuHong Fu 2024. Immunofluorescence protocol for FFPE human post-mortem brain sections to detect alpha-synuclein and tau pathology. protocols.io https://dx.doi.org/10.17504/protocols.io.n92ld8nxxv5b/v2Version created by courtney.wright Wright
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: August 18, 2024
Last Modified: August 22, 2024
Protocol Integer ID: 106180
Keywords: ASAPCRN, human, post-mortem, formalin-fixed, paraffin embedded, alpha-synculein, tau, immunofluorescence
Funders Acknowledgement:
Michael J Fox Foundation
Grant ID: ASAP-000497
Disclaimer
The protocols.io team notes that research involving animals and humans must be conducted according to internationally-accepted standards and should always have prior approval from an Institutional Ethics Committee or Board.
Abstract
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.
Materials
Materials
- HistoChoice‱ Clearing agent - Sigma #H2779
- Series 2 adhesive microscope slides - Trajan, #472042491
- Sodium borohydride - Sigma #71320, Lot #STBJ4218
- Human FC blocker - BD Pharmingen Human BD Fc Block, clone Fc1, #564220
- TrueBlack‱ Background Suppressor- Biotium #23012A
- TrueBlack‱ Lipofuscin Autofluorescence Quencher - Cell Signaling Technology #23007)
- Biotium’s CoverGrip‱ Coverslip Sealant - Biotium #23005)
- EverBrite‱ Mounting Medium - Biotium #23001
- 100%, 95%, 70% ethanol
- 70% formic acid
- 0.1 M sodium citrate buffer pH 6 (house made)
- 0.1 M PBS (house made)
Antibodies
| A | B | C | |
| SNCA (Gt) | R&D, AF1338 | 1:200 | |
| Ubq (Rb) | Abcam, ab7780 | 1:100 | |
| P62 (Ms IgG1) | BD #610833 | 1:100 |
Midbrain and cortical primary antibody details
| A | B | C | |
| Dn anti Gt-AF800 | Thermo, #A32930 | 1:250 | |
| Dn anti Rb-AF647 | Thermo, #A31573 | 1:200 | |
| Dn anti Ms-AF488 | Thermo, #A21202 | 1:200 | |
| Hoescht-405 | Sigma, #B2261 | 1:500 |
Midbrain and cortical secondary antibody details
| A | B | C | D | |
| Midbrain conjugated antibody | ||||
| AT8(Ms, IgG1) | Thermo, #MN1020 | Zenon ™ Ms IgG1 Labeling Kits (AF594) | Thermo, #Z25007 | |
| TH (Rb) | Merck, AB152 | Zenon ™ Rb Labeling Kits (AF532) | Thermo, #Z25303 |
Zenon ™ midbrain conjugated antibody details
| A | B | C | D | |
| Cortical conjugated antibody | ||||
| AT8(Ms, IgG1) | Thermo, #MN1020 | Zenon ™ Ms IgG1 Labeling Kits (AF594) | Thermo, #Z25007 | |
| HuD (Rb) | Abcam ab302514 | Zenon ™ Rb Labeling Kits (AF532) | Thermo, #Z25303 |
Zenon ™ cortical conjugated antibody details
Hardware
- Leica HM325 rotary microtome
- Aptum Bio Retriever 2100, Aptum Biologics Ltd, UK
Safety warnings
For hazard information and safety warnings, please refer to the SDS (Safety Data Sheet) for each of the raw materials used.
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.
Day I - Tissue Prep
Day I - Tissue Prep
- Place slides with FFPE human brain tissues on a slide rack and bake in a 60 °C oven for 01:00:00
1h
De-paraffinize
De-paraffinize
2. Continue using the slide rack, submerge slides in the following solution to de-paraffinize:
a. HistoChoice: 2 x 00:07:00
b. 100% ethanol: 2 x 00:03:00
c. 95% ethanol: 00:03:00
d. 70 % ethanol: 00:03:00
3. Submerge in MiliQ/ultrapure water for 00:03:00 .
4. Tap gently sideways on flat surface covered with paper towels to remove excess water.
19m
Antigen retrieval
Antigen retrieval
5. Submerge slides in 70% Formic Acid for 00:20:00 .
6. Wash in MiliQ/ultrapure water for 2x00:05:00 .
7. Remove slides from the rack and submerge slides directly into 0.01 Molarity (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
of 02:00:00 .
9. Wash with MiliQ/ultrapure water for 00:01:00 , followed by washing with 1 X PBS for 00:05:00 .
2h 31m
Quenching aldehyde group
Quenching aldehyde group
10. Prepare 0.1% Sodium borohydride (NaBH4) in 1xPBS for 00:30:00 of quenching. The solution must always remain chilled in ice.
11. Wash in 1x PBS for 2x00:05:00
35m
Human Fc blocker treatment
Human Fc blocker treatment
12. Add Human FC blocker in 1X PBS with a ratio of 1:50, incubate slides with Human FC blocker at Room temperature for 00:05:00
5m
Background Suppressor system treatment
Background Suppressor system treatment
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 temperature 00:30:00
14. Remove the Background Suppressor and add IF Blocking Buffer (home-made). Room temperature 01:00:00
1h 30m
Primary antibody incubation
Primary antibody incubation
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 48:00:00 in 4 °C
2d
Day 4 - Secondary antibodies
Day 4 - Secondary antibodies
1. Wash slides in PBST 3x00:10:00 . 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 02:00:00
4. Wash slides in PBST 3x00:05:00 , followed by 1x PBS 1x00:05:00
2h 20m
Conjugated antibodies
Conjugated antibodies
5. Prepare conjugated antibodies using Zenon‱ labeling kits within00:30:00 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 02:00:00
7. Wash slides in 1x PBS 3x00:05:00
2h 35m
Post-treatment with TrueBlack® Lipofuscin Autofluorescence Quencher
Post-treatment with TrueBlack® Lipofuscin Autofluorescence Quencher
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 3x00:05:00
5m
Mounting and cover slipping
Mounting and cover slipping
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