Sep 12, 2018

Public workspaceImmunocytochemistry Staining Protocol

 Forked from Immunocytochemistry Staining Protocol
DOI
dx.doi.org/10.17504/protocols.io.thwej7e
  • 1University College Dublin
Simeng Li
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DOI: dx.doi.org/10.17504/protocols.io.thwej7e
External link: http://www.biolegend.com/media_assets/support_protocol/Immunofluorescence_Microscopy_Protocol_050514.pdf
Protocol Citation: Simeng Li 2018. Immunocytochemistry Staining Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.thwej7e
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: In development
We are still developing and optimizing this protocol
Created: September 12, 2018
Last Modified: September 12, 2018
Protocol Integer ID: 15638
Before start
Reagent List:
  • Chamber slides, cover slips, or 12-well plates
  • Phosphate-buffered saline (PBS)
  • Fixation solution: 1% Paraformaldehyde, in PBS
  • Permeabilization solution: 0.5% Triton X-100 in PBS
  • Blocking buffer: 5% FBS in PBS
Sample Preparation
Sample Preparation
Grow cultured cells on cover slips or in wells overnight at 37°C. At the time of fixation, cells should be ~70-80% confluent in single layer.
Rinse cells briefly in PBS.
Fix cells by incubation with freshly made 4% Paraformaldehyde in PBS for 15 minutes at room temperature.
Duration00:15:00
Rinse three times quickly in PBS.
Sample Blocking
Sample Blocking
Block samples in 1 mL of blocking buffer at room temperature for 1 hour.
Duration00:30:00
Sample Staining
Sample Staining
Dilute the primary antibody to the recommended concentration/dilution in blocking buffer.
For 8-well chamber slides, add 200 µL per well. For 12-well plates, add 500 µL per well. Incubate two to three hours at room temperature or overnight at 4°C. If using conjugated antibodies, perform this step in the dark.
For surface staining, rinse 3 times quickly in PBS. 
Duration00:10:00
Prepare fluorochrome-conjugated secondary antibody in blocking buffer according to the manufacturer’s specification data sheet, and add 200 µl per well to the 8-well chamber slides. For 12-well plates, add 500 µL per well.
Incubate the samples for one hour, at room temperature, in the dark.
Duration01:00:00
For surface staining, rinse three times quickly in PBS. 
Duration00:10:00
Counterstain with DAPI for 15 minutes at 37 ℃.
Wash cells 3 times and the third time keep PBS in the wells.
Image CD31 at 488nm and DAPI at 388nm