Single-molecule Immunofluorescence Tissue Staining Protocol for Oligomer Imaging
Rebecca Andrews, Joanne Lachica, Steven F. Lee, Sonia Ghandi
Abstract
This protocol details background fluorescence quenching and immunofluorescence staining of human brain tissue for oligomer imaging.
Attachments
Steps
Immunofluorescences staining protocol for oligomer imaging
Cut 8 µm tissue sections on a microtome and load onto glass slides.
Dry slides 0h 10m 0s at 37°C – cover over the top .
Before staining commences keep slides for a few hours but ideally 0h 10m 0s at 60°C.
De-wax sections through three pots of xylene solution. Use each fresh pots of xylene each time.
De-wax section through pot of xylene solution for 0h 2m 0s. (1/3)
De-wax section through pot of xylene solution for 0h 2m 0s. (/23)
De-wax section through pot of xylene solution for 0h 2m 0s. (3/3)
Take sections through two pots of 100% alcohol.
Take sections through pot of 100% alcohol for 0h 2m 0s. (1/2)
Take sections through pot of 100% alcohol for 0h 2m 0s. (2/2)
Put slides into methanol + hydrogen peroxide (H202) solution (100 ml: 1 ml) for 0h 10m 0s at Room temperature fresh pot each time .
Perform necessary antigen retrieval pretreatments by pressure cooking in citrate buffer.
Pressure cook sections in citrate buffer 6 for 0h 10m 0s at pressure (wait for it to release high pressure air) in cleaned pressure cooker .
Cool under running Milli Q water – never under tap water.
Block non-specific antigen/antibody binding by placing sections in PBS and Goat Serum 10% for 0h 30m 0s. NB Goat Serum is chosen for Goat-Raised antibodies.
Apply primary antibody for 1h 0m 0s at Room temperature.
Wash in PBS with fresh buffer ( at least filtered if not cell culture grade ).
Wash 0h 5m 0s in PBS clean squirty bottle with fresh buffer. (1/3)
Wash 0h 5m 0s in PBS clean squirty bottle with fresh buffer. (23)
Wash 0h 5m 0s in PBS clean squirty bottle with fresh buffer. (3/3)
Apply secondary AlexaFluor antibody for 1h 0m 0s at Room temperature in the dark.
Wash in PBS.
Wash 0h 5m 0s in PBS in the dark. (1/3)
Wash 0h 5m 0s in PBS in the dark. (2/3)
Wash 0h 5m 0s in PBS in the dark. (3/3)
Add filtered (0.22 um) 0.1% Sudan black solution (0.1% sudan black/70% ethanol) for 0h 10m 0s at Room temperature in the dark.
Wash 2-3 times in 30% ethanol.
Mount section with Vectashield and coverslip ( Plasma cleaned slides ).
Take for imaging.
