Feb 23, 2024

Public workspaceSingle-molecule Immunofluorescence Tissue Staining Protocol for Oligomer Imaging V.3

DOI
dx.doi.org/10.17504/protocols.io.5qpvorp6bv4o/v3
  • 1University of Cambridge;
  • 2UCL
Rebecca Andrews
Open access
DOI: dx.doi.org/10.17504/protocols.io.5qpvorp6bv4o/v3
Protocol Citation: Rebecca Andrews, Joanne Lachica, Steven F. Lee, Sonia Ghandi 2024. Single-molecule Immunofluorescence Tissue Staining Protocol for Oligomer Imaging. protocols.io https://dx.doi.org/10.17504/protocols.io.5qpvorp6bv4o/v3Version created by Joseph S Beckwith
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: February 23, 2024
Last Modified: May 31, 2024
Protocol Integer ID: 95660
Keywords: immunofluorescences, oligomer imaging, ASAPCRN
Abstract
This protocol details background fluorescence quenching and immunofluorescence staining of human brain tissue for oligomer imaging.
Attachments
kb3ib25np.pdf
kb3ib25np.pdf
154KB
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.
Materials
Materials and Reagents

  • Microtome
  • Glass slides
  • Xylene solution
  • 100% alcohol
  • Methanol
  • Hydrogen peroxide (H202) solution
  • Citrate buffer pH6
  • Milli Q water
  • Pressure cooker
  • PBS
  • Goat Serum 10%
  • AlexaFluor antibody
  • 0.1% Sudan black solution
  • Vectashield
  • Overslip
Immunofluorescences staining protocol for oligomer imaging
Immunofluorescences staining protocol for oligomer imaging
10m
Cut Thikness8 µm tissue sections on a microtome and load onto glass slides.

Dry slides DurationOvernight at Temperature37 °C – cover over the top.

10m
Overnight
Before staining commences keep slides for a few hours but ideally DurationOvernight at Temperature60 °C .

10m
Overnight
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 Duration00:02:00 . (1/3)
2m
De-wax section through pot of xylene solution for Duration00:02:00 . (/23)
2m
De-wax section through pot of xylene solution for Duration00:02:00 . (3/3)
2m
Take sections through two pots of 100% alcohol.
Note
Use fresh pots each time – methylated spirits.

Take sections through pot of 100% alcohol for Duration00:02:00 . (1/2)

2m
Take sections through pot of 100% alcohol for Duration00:02:00 . (2/2)
2m
Put slides into methanol + hydrogen peroxide (H202) solution (100 ml: 1 ml) for Duration00:10:00 at TemperatureRoom temperature fresh pot each time.
Note
This process will block any staining of endogenous peroxidase in the tissue sections.

10m
Perform necessary antigen retrieval pretreatments by pressure cooking in citrate buffer.
Pressure cook sections in citrate buffer Ph6 for Duration00:10:00 at pressure (wait for it to release high pressure air) in cleaned pressure cooker.

10m
Cool under running Milli Q water – never under tap water.
Critical
Block non-specific antigen/antibody binding by placing sections in PBS and Goat Serum 10% for Duration00:30:00 . NB Goat Serum is chosen for Goat-Raised antibodies.
30m
Apply primary antibody for Duration01:00:00 at TemperatureRoom temperature .

1h
Wash in PBS with fresh buffer (at least filtered if not cell culture grade).
Wash
Wash Duration00:05:00 in PBS clean squirty bottle with fresh buffer. (1/3)

5m
Wash Duration00:05:00 in PBS clean squirty bottle with fresh buffer. (23)
5m
Wash Duration00:05:00 in PBS clean squirty bottle with fresh buffer. (3/3)
5m
Apply secondary AlexaFluor antibody for Duration01:00:00 at TemperatureRoom temperature in the dark.

1h
Wash in PBS.
Wash
Wash Duration00:05:00 in PBS in the dark. (1/3)

5m
Wash Duration00:05:00 in PBS in the dark. (2/3)
5m
Wash Duration00:05:00 in PBS in the dark. (3/3)
5m
Add filtered (0.22 um) 0.1% Sudan black solution (0.1% sudan black/70% ethanol) for Duration00:10:00 at TemperatureRoom temperature in the dark.
10m
Pipetting
Wash 2-3 times in 30% ethanol.
Wash
Mount section with Vectashield and coverslip (Plasma cleaned slides).
Take for imaging.
Imaging