TITLE: Single-molecule Immunofluorescence Tissue Staining Protocol for Oligomer Imaging
AUTHORS: Rebecca Andrews, Joanne Lachica, Steven F. Lee, Sonia Ghandi

[DESCRIPTION]
This protocol details background fluorescence quenching and immunofluorescence staining of human brain tissue for oligomer imaging.

[GUIDELINES]
Use only clean bottles, flasks, magnetic stirrers, tweezers, weighing spatulas, measuring cylinders – everything should be cleaned, dried and covered if left on the side before next use.
Everything should be handled with clean tweezers – gloves should not touch the samples, solutions and ideally anything placed into the solutions where the slides are.

[STEPS]
SECTION: Immunofluorescences staining protocol for oligomer imaging
1. Cut   tissue sections on a microtome and load onto glass slides.

SECTION: Immunofluorescences staining protocol for oligomer imaging
2. Dry slides 10 min at 37 °C – cover over the top.

SECTION: Immunofluorescences staining protocol for oligomer imaging
3. Before staining commences keep slides for a few hours but ideally 10 min at 60 °C.

SECTION: Immunofluorescences staining protocol for oligomer imaging
4. De-wax sections through three pots of xylene solution. Use each fresh pots of xylene each time.
SECTION: Immunofluorescences staining protocol for oligomer imaging
4.1. De-wax section through pot of xylene solution for 2 min. (1/3)
SECTION: Immunofluorescences staining protocol for oligomer imaging
4.2. De-wax section through pot of xylene solution for 2 min. (/23)
SECTION: Immunofluorescences staining protocol for oligomer imaging
4.3. De-wax section through pot of xylene solution for 2 min. (3/3)
SECTION: Immunofluorescences staining protocol for oligomer imaging
5. Take sections through two pots of 100% alcohol. 
 

SECTION: Immunofluorescences staining protocol for oligomer imaging
5.1. Take sections through pot of 100% alcohol for 2 min. (1/2)

SECTION: Immunofluorescences staining protocol for oligomer imaging
5.2. Take sections through pot of 100% alcohol for 2 min. (2/2)
SECTION: Immunofluorescences staining protocol for oligomer imaging
6. Put slides into methanol + hydrogen peroxide (H202) solution (100 ml: 1 ml) for 10 min at Room temperature fresh pot each time.
 

SECTION: Immunofluorescences staining protocol for oligomer imaging
7. Perform necessary antigen retrieval pretreatments by pressure cooking in citrate buffer.
SECTION: Immunofluorescences staining protocol for oligomer imaging
7.1. Pressure cook sections in citrate buffer pH 6 for 10 min at pressure (wait for it to release high pressure air) in cleaned pressure cooker.

SECTION: Immunofluorescences staining protocol for oligomer imaging
7.2. Cool under running Milli Q water – never under tap water.
SECTION: Immunofluorescences staining protocol for oligomer imaging
8. Block non-specific antigen/antibody binding by placing sections in PBS and Goat Serum 10% (taken from the DNA PAINT protocol) for 30 min.
SECTION: Immunofluorescences staining protocol for oligomer imaging
9. Apply primary antibody for 60 min at Room temperature.

SECTION: Immunofluorescences staining protocol for oligomer imaging
10. Wash in PBS with fresh buffer (at least filtered if not cell culture grade).
SECTION: Immunofluorescences staining protocol for oligomer imaging
10.1. Wash 5 min in PBS clean squirty bottle with fresh buffer. (1/3)

SECTION: Immunofluorescences staining protocol for oligomer imaging
10.2. Wash 5 min in PBS clean squirty bottle with fresh buffer. (23)
SECTION: Immunofluorescences staining protocol for oligomer imaging
10.3. Wash 5 min in PBS clean squirty bottle with fresh buffer. (3/3)
SECTION: Immunofluorescences staining protocol for oligomer imaging
11. Apply secondary AlexaFluor antibody for 60 min at Room temperature in the dark.

SECTION: Immunofluorescences staining protocol for oligomer imaging
12. Wash in PBS.
SECTION: Immunofluorescences staining protocol for oligomer imaging
12.1. Wash 5 min in PBS in the dark. (1/3)

SECTION: Immunofluorescences staining protocol for oligomer imaging
12.2. Wash 5 min in PBS in the dark. (2/3)
SECTION: Immunofluorescences staining protocol for oligomer imaging
12.3.   Wash 5 min in PBS in the dark. (3/3)
SECTION: Immunofluorescences staining protocol for oligomer imaging
13. Add filtered (0.22 um) 0.1% Sudan black solution (0.1% sudan black/70% ethanol) for 10 min at Room temperature in the dark.
SECTION: Immunofluorescences staining protocol for oligomer imaging
14. Wash 2-3 times in 30% ethanol.
SECTION: Immunofluorescences staining protocol for oligomer imaging
15. Mount section with Vectashield and coverslip (Plasma cleaned slides).
16. Take for imaging.