Nov 14, 2025

Public workspaceImmunocytochemistry Protocol for Progenitor Cells in 96-Well Plates

DOI
https://dx.doi.org/10.17504/protocols.io.4r3l216jjg1y/v1
  • Guochang Lyu1,
  • Ernest Arenas1
  • 1Karolinska Institute
  • Ernest Arenas: Deceased 15.09.2024
  • SOX6 mDA differentiation
Roberto Garcia Swinburn
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DOI: https://dx.doi.org/10.17504/protocols.io.4r3l216jjg1y/v1
Protocol Citation: Guochang Lyu, Ernest Arenas 2025. Immunocytochemistry Protocol for Progenitor Cells in 96-Well Plates. protocols.io https://dx.doi.org/10.17504/protocols.io.4r3l216jjg1y/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: September 09, 2025
Last Modified: November 14, 2025
Protocol Integer ID: 226843
Keywords: immunocytochemistry protocol for progenitor cell, canonical markers with fluorophore, immunocytochemistry protocol, progenitor cell, canonical marker, fluorophore, plates this protocol
Abstract
This protocol was used to tag canonical markers with fluorophores on in vitro differentiated KOLF2.1J cells.
Troubleshooting
Cell Seeding
Seed ~300,000 progenitor cells per well in a 96-well plate.
Culture as needed before fixation.
Fixation
37m
Remove medium from each well.
2m
Add 4% PFA to each well. Incubate for 20 minutes at 4°C.
20m
Incubation
Wash wells 3× with PBS to remove residual fixative (5 minutes each wash).
15m
Permeabilization & Blocking
1h
Prepare blocking/permeabilization buffer:
  • 5% normal donkey serum
  • 0.3% Triton X-100 in PBS (PBST)
Add buffer to each well. Incubate for 1 hour atTemperatureRoom temperature

1h
Pipetting
Primary Antibody Incubation
16h
Dilute primary antibodies in PBST + 1% donkey serum. Remember to mix antibodies with hosts that will not interact undesiredly with secondary antibodies. Example: goat primary antibody with goat secondary antibodies.
Add to each well. Incubate overnight at 4°C. DurationOvernight Temperature4 °C

16h
Overnight
Temperature
Secondary Antibody Incubation
45m
Wash wells 3× with PBST (5 minutes each wash at room temperature).
15m
Add Alexa Fluor-conjugated secondary antibodies (e.g., Alexa488, Alexa555, or Alexa647) diluted in PBST. Incubate for 30 minutes at room temperature, protected from light. TemperatureRoom temperature Duration00:30:00 . Remember not to mix secondary antibodies that will interact with each other, example goat anti-donkey and donkey anti-rabbit.

30m
Nuclear Staining
15m
Add DAPI to each well. Incubate for 15 minutes at room temperature, protected from light.
15m
Wash 3× with PBS (5 minutes each).
15m
Imaging
Store samples in PBS (short term, <48h) or fluorescence mounting media (mid-long term).
Image using a fluorescence microscope (appropriate channels for Alexa dyes and DAPI).
Imaging