Mar 15, 2024

Public workspaceImmunocytochemistry

DOI
dx.doi.org/10.17504/protocols.io.5qpvor7pdv4o/v1
  • 1Laboratory of Molecular Neurogenetics, Department of Experimental Medical Science, Wallenberg Neuroscience Center and Lund Stem Cell Center, BMC A11, Lund University, 221 84 Lund, Sweden.
Anita Adami
Open access
DOI: dx.doi.org/10.17504/protocols.io.5qpvor7pdv4o/v1
Protocol Citation: anita.adami 2024. Immunocytochemistry. protocols.io https://dx.doi.org/10.17504/protocols.io.5qpvor7pdv4o/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 02, 2023
Last Modified: March 15, 2024
Protocol Integer ID: 76288
Funders Acknowledgement:
Aligning Science Across Parkinson’s through the Michael J. Fox Foundation for Parkinson’s Research
Grant ID: ASAP-000520
Swedish Research Council
Grant ID: 2018-02694
Swedish Brain Foundation
Grant ID: FO2019-0098
Cancerfonden
Grant ID: 190326
Barncancerfonden
Grant ID: PR2017-0053
NIHR Cambridge Biomedical Research Centre
Grant ID: NIHR203312
Swedish Society for Medical Research
Grant ID: S19-0100
National Institutes of Health
Grant ID: HG002385
Swedish Research Council
Grant ID: 2021-03494
Swedish Research Council
Grant ID: 2020-01660
Abstract
This protocol describes how to perform immunocytochemistry on 2D fixed cells
Fixing
Fixing
The cells were washed three times with DPBS (GIBCO) and fixed for 10 minutes with 4% paraformaldehyde (Merck Millipore), followed by three more rinses with DPBS.
Blocking and permeabilisation
Blocking and permeabilisation
1h
The cells were blocked for Duration01:00:00 in blocking solution (KPBS 0.25% Triton X-100 (Fisher Scientific) and 5% normal donkey serum) at TemperatureRoom temperature .
1h
Staining
Staining
2h
The primary antibody was added to the blocking solution and incubated DurationOvernight at TemperatureRoom temperature (follow manufacturer's instructions for dilutions).
1h
The cells were then washed the next day three times with KPBS 0.25%.
The secondary antibody was added with DAPI (1:1000; Sigma-Aldrich) to the blocking solution and incubated at TemperatureRoom temperature for Duration01:00:00 .

1h
The cells were finally rinsed 2-3 times with KPBS and visualised on a Leica microscope (model DMI6000B).