Feb 16, 2024

Public workspaceImmunofluorescence staining, vibratome sections

DOI
dx.doi.org/10.17504/protocols.io.3byl4qoxrvo5/v1
  • 1Katholieke Universiteit Leuven
Eduard Bentea
Open access
DOI: dx.doi.org/10.17504/protocols.io.3byl4qoxrvo5/v1
Protocol Citation: Eduard Bentea, María Sanchiz Calvo, Veerle Baekelandt 2024. Immunofluorescence staining, vibratome sections. protocols.io https://dx.doi.org/10.17504/protocols.io.3byl4qoxrvo5/v1
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 16, 2024
Last Modified: May 31, 2024
Protocol Integer ID: 95341
Keywords: ASAPCRN
Funders Acknowledgement:
ASAP (Aligning Science Across Parkinson's)
Abstract
Protocol for performing immunofluorescence staining on free-floating vibratome cut brain sections from rats or mice.
Materials
Antigen retrieval solution (10 mM citrate buffer pH 6.0)

Prepare stock solutions:
Solution A : 0.1 M citric acid solution
Solution B : 0.1 M sodium citrate solution

Prepare working solution:
Add 9 mL of Stock solution A and 41 mL of Stock solution B to 400 mL of AD
Adjust pH to 6.0
Fill until 500 mL with AD
Day 1
Day 1
2h
Transfer sections to be stained to a new 24-well plate filled with 1X PBS. All incubations are with Amount500 µL solution per well unless stated otherwise.

1X PBS rinse.
Antigen retrieval step (using oven).
Incubate sections in antigen retrieval solution (10 mM citrate buffer pH 6.0 ; see recipe in Materials) at 80oC for Duration00:30:00 .

30m
Place the well plate in the same buffer on ice for Duration00:20:00 .

20m
1X PBS rinse.
Wash sections in 1X PBS for Duration00:05:00 at room temperature on wobbler.

5m
Wash sections in 1X PBS for Duration00:05:00 at room temperature on wobbler.
5m
Incubate sections with Amount250 µL blocking solution (PBS-T + 10% donkey serum) per well for Duration01:00:00 at room temperature on wobbler. (PBS-T = PBS + 0.1% Tergitol)

1h
Dilute primary antibodies at the required concentration in PBS-T + 10% donkey serum.
Incubate sections with Amount250 µL primary antibodies overnight at room temperature.

Day 2
Day 2
2h
1X PBS-T rinse.
Wash sections in 1X PBS-T for Duration00:05:00 at room temperature on wobbler.
5m
Wash sections in 1X PBS-T for Duration00:05:00 at room temperature on wobbler.
5m
Dilute secondary antibodies at the required concentration in PBS-T.
Incubate sections with Amount250 µL secondary antibodies for Duration02:00:00 at room temperature (in the dark).

2h
1X PBS rinse.
Wash sections in 1X PBS for Duration00:05:00 at room temperature on wobbler.
5m
Wash sections in 1X PBS for Duration00:05:00 at room temperature on wobbler.
5m
Briefly rinse sections in 1/2 PBS + 1/2 AD and allow to dry.
Mount with Mowiol.