Feb 13, 2026

Public workspaceImmunofluorescence (IF) staining of MTSs on MWC-chips

DOI
https://dx.doi.org/10.17504/protocols.io.6qpvry262gmk/v1
  • Zhe Zhao1
  • 1CAS Key Laboratory of Nano-Bio Interface, Suzhou Institute of Nano-Tech and Nano-Bionics, Chinese Academy of Sciences, Jiangsu, 215123, China.
  • MTS
Guangli Suo
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DOI: https://dx.doi.org/10.17504/protocols.io.6qpvry262gmk/v1
Protocol Citation: Zhe Zhao 2026. Immunofluorescence (IF) staining of MTSs on MWC-chips. protocols.io https://dx.doi.org/10.17504/protocols.io.6qpvry262gmk/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 13, 2026
Last Modified: February 13, 2026
Protocol Integer ID: 243182
Keywords: chips immunofluorescence, mtss on mwc, mtss, mwc
Funders Acknowledgements:
National Natural Science Foundation of China
Grant ID: 82202345
Abstract
Immunofluorescence (IF) staining of MTSs on MWC-chips (CD44,ALDH1A1)
Materials
4% paraformaldehyde (Beyotime), 1% Triton-X 100 in PBS, 10% goat serum, primary antibodies against ALDH1A1 (Alexa594-conjugated, Abcam), CD44 (Alexa488-conjugated, Abcam), FAP (Alexa647-conjugated, RD System), Hoechst 33342 dye, confocal microscopy (OLYMPUS), ImageJ software.
Troubleshooting
Immunofluorescence (IF) staining of MTSs on MWC-chips
Once MTSs were formed after 5 days of culture, the SC-Medium was aspirated from the culture dish, and the MTSs in microwells were fixed with 4% paraformaldehyde (Beyotime) for 20 minutes.
Subsequently, they were washed with PBS.
The MTSs were then permeabilized with 1% Triton-X 100 in PBS for 30 minutes and blocked with 10% goat serum for another 30 minutes.
Next, cells were incubated overnight at 4°C with primary antibodies against ALDH1A1 (Alexa594-conjugated, Abcam), CD44 (Alexa488-conjugated, Abcam), and FAP (Alexa647-conjugated, RD System).
Nuclei were stained using Hoechst 33342 dye.
Images were captured using confocal microscopy (OLYMPUS) and were analyzed using ImageJ software.