Feb 23, 2026
Immunofluorescence under confinement
- Ankita Jha1
- 1National Institutes of Health
Protocol Citation: Ankita Jha 2026. Immunofluorescence under confinement. protocols.io https://dx.doi.org/10.17504/protocols.io.n2bvjn48ngk5/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 26, 2024
Last Modified: February 23, 2026
Protocol Integer ID: 108415
Keywords: immunofluorescence under confinement cell, agarose confinement, immunofluorescence, confinement cell, desired antibody
Abstract
Cells under agarose confinement were fixed and labelled with desired antibodies.
Troubleshooting
Immunofluorescence under confinement
5h 20m
Samples under agarose pad confinement were fixed with 4% paraformaldehyde (PFA; Electron Microscopy Sciences, 15710) in cytoskeleton buffer (CB; 10 mM MES, 3 mM MgCl₂, 138 mM KCl, 2 mM EGTA) for 20 min at 37 °C, after which the agarose pad was gently removed.
20m
Cells were permeabilized with 0.5% Triton X-100 in CB for 5 min at room temperature (RT) and quenched with 10 mM glycine in CB for 5 min.
10m
Samples were washed with Tris-buffered saline (TBS; 20 mM Tris, pH 7.6, 137 mM NaCl) (2 × 5 min, then 2 × 10 min) and blocked for 1 h at RT in blocking buffer (2% IgG- and protease-free BSA and 0.1% Tween-20 in TBS).
1h 30m
Cells were incubated for 2h at RT or overnight at 4 °C with primary antibodies against CD44 (BJ18;
BioLegend), Ezrin (3C12; Invitrogen), EGFR (H11; Invitrogen), or phospho-ERM
(Thr567/564/558; Cell Signaling Technology) diluted in TBS.
2h
Following three TBS washes, samples were incubated for 1 h at RT with
fluorophore-conjugated secondary antibodies (1:500; Jackson ImmunoResearch) and
Alexa Fluor 647–phalloidin (1:200; Thermo Fisher Scientific) diluted in
blocking buffer.
1h
Samples were washed with TBS (2 × 10 min) and mounted in Dako
mounting medium (S3023) on glass-bottom dishes.
20m