Jan 31, 2024
Immunofluorescence on human brain FFPE sections to identify neuromelanin in A6, A9, and A10
- Hongyun Li1,2,
- YuHong Fu1,2
- 11Brain and Mind Centre & Faculty of Medicine and Health School of Medical Sciences, The University of Sydney, Sydney, NSW 2050, Australia;
- 22Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD, 20815
Protocol Citation: Hongyun Li, YuHong Fu 2024. Immunofluorescence on human brain FFPE sections to identify neuromelanin in A6, A9, and A10. protocols.io https://dx.doi.org/10.17504/protocols.io.81wgbxejolpk/v1
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: January 31, 2024
Last Modified: May 31, 2024
Protocol Integer ID: 94433
Keywords: ASAPCRN, IHC, NM, A6, A9, A10, alpha-synculein, FFPE
Funders Acknowledgement:
The Michael J. Fox Foundation for Parkinson’s Research (MJFF) and the Aligning Science Across Parkinson’s (ASAP) initiative
Grant ID: ASAP-020505
Abstract
This is the protocol to scan brain neuromelanin with reduced impact of the chemicals on their original colour and intensity. The immunofluorescence staining protocol is designed to identify human A6, A9, and A10 cell clusters and alpha-synuclein pathology.
Preparation
Preparation
1h 10m
Select sections, label the staining method on slides, and load slides to slide racks.
1h
Check the medium and reagents ready for use.
10m
Deparaffin and rehydration
Deparaffin and rehydration
1h 33m
Bake the slides in a 60 °C oven for one hour.
1h
Dip slides in HistoChoice for 2 x 7 minutes.
14m
Dip slides in 100% ethanol for 2 x 3 minutes.
6m
Dip slides in 95% ethanol for 3 minutes.
3m
Dip slides in 70 % ethanol for 3 minutes.
3m
Dip slides in MQ water for 3 minutes.
3m
Dip slides in 1XPBS.
Note: The slides can be mounted with DAKO fluorescence mounting medium (Agilent, cat# S302380-2) for brightfield scanning by VS200 to acquire neuromelanin images, then de-coverslip in 1XPBS to proceed for the following antigen retrieval step.
1m
Heat-induced Antigen retrieval
Heat-induced Antigen retrieval
2h 6m
Transfer slides into the slides chamber filled with 1X citric buffer (CB) pH6.0, and apply HIAR using a programmable antigen retrieval cooker (Aptum Bio Retriever 2100, Aptum Biologics Ltd, UK) at a peak temperature of 121 °C , followed by the gradual cooling procedure for ~2 hours.
2h
Dip slides in MQ water for 1 minute.
1m
Dip slides in 1XPBS for 5minutes
5m
Quench aldehyde group
Quench aldehyde group
50m
Prepare NaBH4 (Sigma #72320, stored in the toxic cabinet) at a final concentration of 0.5% (w/v) in 1X PBS. Make this fresh in the fume hood!
10m
Transfer slides into this quenching buffer for 30 minutes (on ice).
30m
Slides undergone PBS washing 2 X 5 minutes.
10m
Suppressing lipofuscin autofluorescence
Suppressing lipofuscin autofluorescence
43m
Immersing slides in 70% ethanol for ~1 minute.
1m
Transfer the slides into 0.1% SBB (in 70% ethanol) for ~30 minutes.
30m
Wash slide with 70% ethanol for ~1 minute (dip several times)
1m
Rinsing slides with pure H2O briefly.
1m
Wash slides in 1XPBS 2 X 5 minutes.
10m
Blocking buffer treatment
Blocking buffer treatment
2h
Set up the StainTray slide staining system (Sigma, Z670146) or incubation boxes. Circle the samples with PAP pen.
30m
Blocking with Human Fc blocker: Human FC blocker (BD Pharmingen Human BD Fc Block, clone Fc1, #564220) in 1XPBS 1:200 @ Room temperature for 30 minutes.
30m
Blocking with IF buffer @ Room temperature for 60 minutes.
1h
Primary antibody incubation for indirect labeling
Primary antibody incubation for indirect labeling
12h 20m
Make a Primary antibody cocktail in IF buffer.
| A | B | |
| CalB1(Ch) | Antibodies.com #A85369 | |
| ALDH1A1 (Gt) | R&D AF5869 |
Primary Antibodies
20m
Incubate sections with primary antibody cocktail in the CoolRm @ 4 °C overnight.
12h
Secondary antibodies incubation (from this step, protect from light)
Secondary antibodies incubation (from this step, protect from light)
2h 30m
Wash sections with 1xPBS 3X10 minutes
30m
Make the 2nd antibody cocktails as above in IF blocking buffer @ Room temperature for 120 minutes.
| A | B | C | D | |
| Cat# | ThermoFisher SA5-10091 | Sigma SAB4600031 | Sigma B2261 | |
| Reagent | Dn@Gt-DL755 | Dn@Ch IgY-CF488A | Hoechst 33,342 (1mg/ml stock) | |
| Dilution | 1:200 | 1:250 | 1:1000 |
Reagent list
2h
Direct labelling with conjugated antibodies
Direct labelling with conjugated antibodies
2h 30m
Wash sections in 1XPBS 3X5 minutes.
15m
Dilute conjugated antibodies into IF blocking buffer as below @ Room temperature for 120 minutes.
| A | B | C | |
| Conjugated Abs | TH- AF647 | S129-AF568 | |
| Cat# | BioLegend #818008 | Abcam Ab307166 | |
| Dilution | 1:100 | 1:100 |
Conjugated antibodies
2h
Wash sections in 1XPBS 3X5 minutes.
15m
Coverslip
Coverslip
1h 30m
Leave sections at dark for drying for about 5~10 minutes or dry all the solution residue on the sections, mounting with anti-fade media (DAKO Fluorescence Mounting Medium), and leave @ Room temperature for >30 minutes.
1h
Seal the corners of the coverslips with nail polish.
30m
Storage before scan
Storage before scan
10m
Store slides in the slide box and store in the fridge or cool Rm.
10m
Ready for VS200 scanning.
Appendix of medium
Appendix of medium
Home-made IF Blocking buffer (NDS):
• 2% Donkey serum (Sigma, D9663)
• 1% BSA (best with IgG-free and protease-free) (Sigma, A9085 or JIR #001-000-173).
• 0.2% TritonX-100 (Sigma, T9284).
• 0.1% gelatine (from fish skin, Sigma, G7041).
• 0.1% Tween-20 (Sigma, P1379).
• 0.01% Sodium Azide (Sigma, S2002)
in 1XPBS, aliquoted and stored @ -20 °C .