TITLE: Immunofluorescence on human brain FFPE sections to identify neuromelanin in A6, A9, and A10
AUTHORS: Hongyun Li, YuHong Fu

[DESCRIPTION]
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]
SECTION: Preparation
1. Select sections, label the staining method on slides, and load slides to slide racks.

SECTION: Preparation
2. Check the medium and reagents ready for use.
SECTION: Deparaffin and rehydration
3. Bake the slides in a 60 °C  oven for one hour.

SECTION: Deparaffin and rehydration
4. Dip slides in HistoChoice for 2 x 7 minutes.

SECTION: Deparaffin and rehydration
5. Dip slides in 100% ethanol for 2 x 3 minutes.

SECTION: Deparaffin and rehydration
6. Dip slides in  95% ethanol for 3 minutes.

SECTION: Deparaffin and rehydration
7. Dip slides in 70 % ethanol for 3 minutes.

SECTION: Deparaffin and rehydration
8. Dip slides in MQ water for 3 minutes. 

SECTION: Deparaffin and rehydration
9. 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.
SECTION: Heat-induced Antigen retrieval
10. 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.

SECTION: Heat-induced Antigen retrieval
11. Dip slides in MQ water for 1 minute. 

SECTION: Heat-induced Antigen retrieval
12. Dip slides in 1XPBS for 5minutes
SECTION: Quench aldehyde group
13. 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!

SECTION: Quench aldehyde group
14. Transfer slides into this quenching buffer for 30 minutes (on ice).  

SECTION: Quench aldehyde group
15. Slides undergone PBS washing 2 X 5 minutes. 
SECTION: Suppressing lipofuscin autofluorescence
16. Immersing slides in 70% ethanol for ~1 minute.  

SECTION: Suppressing lipofuscin autofluorescence
17. Transfer the slides into 0.1% SBB (in 70% ethanol) for ~30 minutes.   

SECTION: Suppressing lipofuscin autofluorescence
18. Wash slide with 70% ethanol for ~1 minute (dip several times)

SECTION: Suppressing lipofuscin autofluorescence
19. Rinsing slides with pure H2O briefly.
SECTION: Suppressing lipofuscin autofluorescence
20. Wash slides in 1XPBS 2 X 5 minutes.  
SECTION: Blocking buffer treatment
21. Set up the StainTray slide staining system (Sigma, Z670146) or incubation boxes. Circle the samples with PAP pen.
SECTION: Blocking buffer treatment
22. 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.

SECTION: Blocking buffer treatment
23. Blocking with IF buffer @ Room temperature for 60 minutes.
SECTION: Primary antibody incubation for indirect labeling
24. Make a Primary antibody cocktail in IF buffer.
 
 
 
SECTION: Primary antibody incubation for indirect labeling
25. Incubate sections with primary antibody cocktail in the CoolRm @ 4 °C  overnight.

SECTION: Secondary antibodies incubation (from this step, protect from light)
26. Wash sections with 1xPBS 3X10 minutes

SECTION: Secondary antibodies incubation (from this step, protect from light)
27. Make the 2nd antibody cocktails as above in IF blocking buffer @ Room temperature for 120 minutes.
 
 
 
SECTION: Direct labelling with conjugated antibodies
28. Wash sections in 1XPBS 3X5 minutes.

SECTION: Direct labelling with conjugated antibodies
29. Dilute conjugated antibodies into IF blocking buffer as below @ Room temperature for 120 minutes.
 
 
 
SECTION: Direct labelling with conjugated antibodies
30. Wash sections in 1XPBS 3X5 minutes.
SECTION: Coverslip
31. 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.

SECTION: Coverslip
32. Seal the corners of the coverslips with nail polish.  
SECTION: Storage before scan
33. Store slides in the slide box and store in the fridge or cool Rm.

SECTION: Storage before scan
34. Ready for VS200 scanning. 
SECTION: Appendix of medium
35. 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.