Mar 15, 2024
Immunohistochemistry (neural organoids)
- 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.
Protocol Citation: anita.adami 2024. Immunohistochemistry (neural organoids). protocols.io https://dx.doi.org/10.17504/protocols.io.n92ldp22nl5b/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 03, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 76335
Keywords: ASAPCRN
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 protocols describes how to perform immunohistochemistry on neural organoids
Fixing and mounting
Fixing and mounting
4h
The organoids were fixed in 4% paraformaldehyde for 02:00:00 at Room temperature .
2h
They were subsequently washed three times with KPBS and left in a 1:1 OCT:30% sucrose solution and OCT (HistoLab) mixture Overnight .
2h
The organoids were then transferred to a cryomold containing OCT and frozen on dry ice and stored at -80°C.
Sectioning
Sectioning
Prior to the staining protocol, the organoids were cryosectioned at a thickness of 20 μM.
Staining
Staining
2h 10m
For immunohistochemistry, sections were washed in PBS1X for 00:10:00 and then blocked and permeabilized in Blocking solution (0.3% Triton X-100 and 4% normal donkey serum in PBS1X) for at least 01:00:00 .
1h 10m
The sections were then incubated with primary antibodies in blocking solution at 4 °C Overnight .
The primary antibodies used for neural organoids characterisation were rabbit anti-PAX6 (1:300; BioLegend, Cat# 3700, RRID:AB_2242334)) and rat anti-ZO1 NB110-68140, RRID:AB_1111431(1:300; Novus)).
1h
After incubation with the primary antibodies, the sections were incubated for 1 h with the appropriate secondary antibodies (Alexa Fluor 488, 594, 647 used at 1:400; Molecular Probes).
Mounting
Mounting
Finally, the sections were mounted on gelatin-coated slides and coverslipped with PVA-DABCO containing DAPI (1:1000).