Sep 09, 2022

Public workspaceImmunocytochemistry (ICC)

DOI
dx.doi.org/10.17504/protocols.io.q26g74w79gwz/v1
  • 1Queen Square UCL Institute of Neurology;
  • 2The Francis Crick Institute
Minee L Choi
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DOI: dx.doi.org/10.17504/protocols.io.q26g74w79gwz/v1
Protocol Citation: mineechoi 2022. Immunocytochemistry (ICC). protocols.io https://dx.doi.org/10.17504/protocols.io.q26g74w79gwz/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: May 25, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 63207
Keywords: ASAPCRN
Abstract
This protocol describes how to do immunocytostochemistry for primary and hiPSC-derived cells.
Cell fixation
Cell fixation
Cells are fixed in Concentration4 % volume paraformaldehyde (PFA) and stored in phosphate-buffered saline (PBS) until use.





Permeabilizing and blocking cells
Permeabilizing and blocking cells
Wash cells with PBS twice.
Incubate the cells in Concentration0.2 % volume Triton X-100, Concentration5 % volume bovine serum albumin (BSA) for Duration01:00:00 atTemperatureRoom temperature .
Note
Concentration5 % volume BSA (made in PBS) is used to block non-specific binding.



Note
For ATTO 425 labelled Aptamer staining, cells are permeabilized with Concentration0.25 % volume Triton X-100 and blocked with Concentration10 % volume normal goat serum (NGS) for Duration00:20:00 followed by another Duration03:00:00 with Concentration0.1 % volume Trion X-100 and Concentration10 % volume NGS.




1h
Incubate cells in primary antibodies
Incubate cells in primary antibodies

Note
Do not wash after permeabilising and blocking steps

Dilute primary antibody in Concentration5 % volume BSA and incubate cells at Temperature4 °C , DurationOvernight or Duration01:00:00 at TemperatureRoom temperature .
Note
The final volume should be sufficient to cover each coverslip around Amount170 µL for 8-ibid chambers, #80806). For 8-ibid, it recommends incubating the cells at room temperature for Duration01:00:00 at TemperatureRoom temperature if possible.


2h
Wash cells with Concentration5 % volume BSA for Duration00:05:00 three times.

5m
Incubate cells in secondary antibodies
Incubate cells in secondary antibodies
1h 5m
1h 5m
Dilute primary antibody in Concentration5 % volume BSA and incubate cells at Temperature4 °C DurationOvernight or Duration01:00:00 at TemperatureRoom temperature .

1h 5m
Wash cells with Concentration5 % volume BSA for Duration00:05:00 three times away from light.
Note
Add Hoechst (Concentration10 micromolar (µM) ) in the second wash and leave for Duration00:15:00 .



5m
Take away PBS and load anti-fading medium to cover cells.
Note
For the short term, imaging in PBS is also fine.