Sep 29, 2025

Public workspaceImmunocytochemistry

DOI
https://dx.doi.org/10.17504/protocols.io.yxmvm3146l3p/v1
  • Oriol Busquets1,2,
  • Hanqin Li3,2,
  • Khaja Mohieddin Syed3,2,
  • Riana Lo Bu1,2,
  • Frank Soldner1,2,
  • Dirk Hockemeyer3,2
  • 1Albert Einstein College of Medicine, 1301 Morris Park Ave., Bronx, NY 10461, USA.;
  • 2Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD, 20815.;
  • 3University of California, Berkeley, Berkeley, CA 94720, USA
Oriol Busquets
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DOI: https://dx.doi.org/10.17504/protocols.io.yxmvm3146l3p/v1
Protocol Citation: Oriol Busquets, Hanqin Li, Khaja Mohieddin Syed, Riana Lo Bu, Frank Soldner, Dirk Hockemeyer 2025. Immunocytochemistry. protocols.io https://dx.doi.org/10.17504/protocols.io.yxmvm3146l3p/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: January 09, 2024
Last Modified: September 29, 2025
Protocol Integer ID: 93212
Keywords: ASAPCRN, immunocytochemistry this protocol, immunocytochemistry, protocol overview, protocol, differentiated culture
Funders Acknowledgements:
Aligning Science Across Parkinson’s (ASAP)
Grant ID: ASAP-000486
Aligning Science Across Parkinson’s (ASAP)
Grant ID: ASAP-024409
Abstract
This protocol summarizes the steps to immunostain hPSCs or differentiated cultures.

Protocol Overview
A. Cell fixation
B. Immunocytochemistry
Attachments
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Immunocytochemistry....
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Materials
Reagent table:
ItemVendorCatalog NumberDilution
Paraformaldehyde 32%EMS15714-
DPBS (No calcium, No magnesium)ThermoFisherSH30028-
Triton X-100Sigma-
Normal Donkey SerumMillipore530-100-
Bovine Serum Albumin (BSA)FisherBP1600-
Anti-FOXA2R&D SystemsAF24001:500
Anti-THPelFreezeP60101!:250
Anti-PU.1Cell SIgnaling Tech22581:300
Anti-Iba1Abcam50761:800
Anti-CX3CR1Biolegend3416021:100
Anti-P2RY12SigmaHPA0145181:300
Donkey anti-Mouse IgG (HL) Highly Cross Adsorbed Secondary Antibody Alexa Fluor 488InvitrogenA-212061:500
Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor‱ 555InvitrogenA-315721:200
Donkey anti-Rabbit IgG (HL) Highly Cross Adsorbed Secondary Antibody Alexa Fluor 594InvitrogenA-212071:500
Donkey anti-Rat IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor‱ 647InvitrogenA-789471:200
DAPIThermoFisherD13061 ug/ml final
ProLong‱ Glass Antifade MountantThermoFisherP36984-

Troubleshooting
Cell fixation

Note
Cell cultures can be imaged directly after staining in the same plate they were cultured in. For higher quality images, cells should be cultured on coverslips and then mounted onto microscope slides after staining.

Note
At this stage do not shake the plate to avoid cell detachment.

Remove the media and gently rinse the cells with 1X PBS. Remove PBS and add 4% PFA in PBS. Incubate the cells at TemperatureRoom temperature (RT) for Duration00:15:00 to 20 minutes.
15m
After fixation, discard the PFA and wash cells with 1xPBS at TemperatureRoom temperature for 3 times Duration00:10:00 each.
10m
At this stage, samples may be stored at Temperature4 °C for up to a week before continuing with the staining process. It is recommended to seal with parafilm to prevent evaporation and store the plate in the dark.
Immunocytochemistry
4h 20m

Note
Gentle agitation with an orbital shaker may be used after fixation during the washing and staining steps.

Note
Cell permeabilization is required for immunostaining of intracellular proteins and optional for immunostaining of surface proteins using antibodies known to recognize extracellular epitopes.

Permeabilization (optional for surface proteins): add 0.3% Triton X-100 in PBS and incubate the cells for Duration00:15:00 to 20 minutes at TemperatureRoom temperature .
15m
Wash the cells three times with 1xPBS at room temperature for Duration00:10:00 each.
10m
Blocking: Add blocking solution (10% serum or 3% BSA in PBS) and incubate for at least Duration01:00:00 at TemperatureRoom temperature . Note: source/species of serum used in blocking solution must be compatible with host species of the primary antibody.
1h
Remove the blocking solution and add the primary antibody solution. Primary antibody/ies are diluted in blocking solution to the appropriate concentration.
Incubate the plate at Temperature4 °C DurationOvernight .
1h
Remove the primary antibody solution and wash the cells with 1xPBS for three times Duration00:10:00 each at TemperatureRoom temperature .
10m
Add the secondary antibody/ies diluted in PBS to the appropriate concentration. Incubate for Duration01:00:00 at TemperatureRoom temperature in the dark. Note: all subsequent steps after the addition of the secondary antibody should be performed under limited light exposure of samples to light to prevent fluorescence bleaching.
1h
Remove the secondary antibody solution and wash the cells with 1xPBS for three times Duration00:10:00 each at TemperatureRoom temperature .
10m
Add DAPI diluted in 1xPBS and incubate the cells at RT for Duration00:05:00 to 10 minutes.
5m
Rinse the cells three times with 1xPBS. Samples can be stored in the fridge until they are imaged directly from the plate or mounted onto microscope slides for imaging.
Mounting: add a drop of ProLong‱ Glass Antifade Mountant on a microscope slide and place the coverslip on top avoiding bubble formation. Keep in the dark until mountant has dried and image directly or store in the fridge until use.