Immunofluorescence on human brain FFPE sections to identify neuromelanin in A6, A9, and A10
YuHong Fu, Hongyun Li
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.
Steps
Preparation
Select sections, label the staining method on slides, and load slides to slide racks.
Check the medium and reagents ready for use.
Deparaffin and rehydration
Bake the slides in a 60°C oven for one hour.
Dip slides in HistoChoice for 2 x 7 minutes.
Dip slides in 100% ethanol for 2 x 3 minutes.
Dip slides in 95% ethanol for 3 minutes.
Dip slides in 70 % ethanol for 3 minutes.
Dip slides in MQ water for 3 minutes.
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.
Heat-induced Antigen retrieval
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.
Dip slides in MQ water for 1 minute.
Dip slides in 1XPBS for 5minutes
Quench aldehyde group
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!
Transfer slides into this quenching buffer for 30 minutes (on ice).
Slides undergone PBS washing 2 X 5 minutes.
Suppressing lipofuscin autofluorescence
Immersing slides in 70% ethanol for ~1 minute.
Transfer the slides into 0.1% SBB (in 70% ethanol) for ~30 minutes.
Wash slide with 70% ethanol for ~1 minute (dip several times)
Rinsing slides with pure H2O briefly.
Wash slides in 1XPBS 2 X 5 minutes.
Blocking buffer treatment
Set up the StainTray slide staining system (Sigma, Z670146) or incubation boxes. Circle the samples with PAP pen.
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.
Blocking with IF buffer @ Room temperature for 60 minutes.
Primary antibody incubation for indirect labeling
Make a Primary antibody cocktail in IF buffer.
| A | B |
|---|---|
| CalB1(Ch) | Antibodies.com #A85369 |
| ALDH1A1 (Gt) | R&D AF5869 |
Primary Antibodies
Incubate sections with primary antibody cocktail in the CoolRm @ 4°C overnight.
Secondary antibodies incubation (from this step, protect from light)
Wash sections with 1xPBS 3X10 minutes
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
Direct labelling with conjugated antibodies
Wash sections in 1XPBS 3X5 minutes.
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
Wash sections in 1XPBS 3X5 minutes.
Coverslip
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.
Seal the corners of the coverslips with nail polish.
Storage before scan
Store slides in the slide box and store in the fridge or cool Rm.
Ready for VS200 scanning.
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.
