Feb 19, 2025
Immunohistochemistry (Brain organoids)
- 1Lund University
External link: https://doi.org/10.1101/2025.01.17.633315
Protocol Citation: Anita Adami 2025. Immunohistochemistry (Brain organoids). protocols.io https://dx.doi.org/10.17504/protocols.io.261gerkm7l47/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 14, 2025
Last Modified: February 19, 2025
Protocol Integer ID: 120306
Keywords: ASAPCRN
Funders Acknowledgements:
Aligning Science Across Parkinson’s through the Michael J. Fox Foundation for Parkinson’s Research
Grant ID: ASAP-000520
Aligning Science Across Parkinson’s through the Michael J. Fox Foundation for Parkinson’s Research
Grant ID: ASAP-024296
Aligning Science Across Parkinson’s through the Michael J. Fox Foundation for Parkinson’s Research
Grant ID: ASAP-025170
Swedish Research Council
Grant ID: 2018-02694
Swedish Brain Foundation
Grant ID: FO2019-0098
Cancerfonden
Grant ID: 190326
Barncancerfonden
Grant ID: PR2017-0053
Abstract
This protocol describes how to prepare brain organoids slides and how to perform immunostainings to study the expression and localization of target proteins in this 3D model.
Sample Preparation
Sample Preparation
4h
4h
Organoids collected for staining were transferred into a 24-well plate and fixed with 4%
paraformaldehyde (PFA) for02:00:00 .
2h
Fixed organoids were then rinsed twice in DPBS and immersed in a 1:1 OCT (catalog no. 45830, HistoLab) and 30% sucrose emulsion in which they were incubated Overnight at 4 °C on gentle shake.
2h
On the following day, the organoids were transferred to a cryomold in OCT and frozen on dry ice, to be stored at -80 °C until cryosectioning.
Organoids were cut on a cryostat at -20 °C into sections of 20 um thickness and placed onto Superfrost Plus microscope slides.
Immunohistochemistry
Immunohistochemistry
3h 30m
3h 30m
A hydrophobic PAP Pen was used to draw the borders of the slides.
Slides were then washed twice in 1X TKPBS (PBS + 0.3% Triton X-100) and fixed again with 4% PFA for 00:10:00 .
10m
After two washes with KPBS for 00:10:00 , slides were immersed in Blocking solution (TKPBS + 5% Normal Donkey Serum) for at least 01:00:00 .
1h 10m
300 ul of Primary antibody solution (blocking solution+ primary antibodies at the chosen dilution) were added to each slide and these were incubated Overnight at 4 °C in a humidified chamber.
1h
On the following day, slides were washed three times with KPBS for 00:10:00
10m
300 ul of Secondary antibody solution (blocking solution + secondary antibodies at the chosen dilution, 1:1000 DAPI) were added to each slide and these were incubated or at least 01:00:00 at Room temperature protected from light.
1h
Slides were then washed three times with KPBS for 00:10:00
Slides were mounted with FluorSave mounting medium, allowed to dry at Room temperature and then stored at 4 °C until imaged.
Slides were imaged on a confocal Microscope Leica TSC SP8 and images were cropped and adjusted on ImageJ Fiji.