Feb 19, 2019
Version 3
Mammalian Cell Staining V.3
- 1University of Arizona
- 481b Laboratory
Protocol Citation: Kenneth Schackart, Kattika Kaarj 2019. Mammalian Cell Staining. protocols.io https://dx.doi.org/10.17504/protocols.io.x95fr86
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 18, 2019
Last Modified: February 19, 2019
Protocol Integer ID: 20509
Abstract
This protocol details how to stain mammalian cells cultured on a 96-well plate. Actin filaments, focal adhesion sites (as indicated by the presence of vinculin), and nuclei will be stained.
Materials
- 4% Paraformaldehyde solution
- 0.1% Triton X-100 solution in PBS
- Blocking buffer (PBS + 1% bovine serum albumin)
- Washing buffer (PBS + 0.05% Tween-20)
- Anti-vinculin solution (1:500 in blocking buffer)
- TRITC-conjugated phalloidin and FITC-conjugated antivinculin secondary antibody solution (1:1:248, TRITC:FITC:blocking buffer) referred to as FITC:TRITC
- DAPI solution (1:249 DAPI:blocking buffer)
- Phosphate buffered saline (PBS)
Fix the cells
Fix the cells
Remove cell culture media.
Add 100 µL of 4 % volume paraformaldehyde solution.
Incubate for 00:05:00 .
Perforate cell membrane
Perforate cell membrane
Remove paraformaldehyde solution.
Wash twice with 100 µL washing buffer.
Note
Washing buffer is PBS with the detergent Tween-20.
Add 100 µL of 0.1 % volume Triton X-100.
Incubate for 00:05:00 .
Block unspecific binding
Block unspecific binding
Remove Triton X-100.
Wash twice with 100 µL washing buffer.
Add 100 µL blocking buffer.
Note
Blocking buffer is PBS with BSA (bovine serum albumin) and is used to prevent unspecific binding.
Incubate for00:10:00 .
Bind anti-vinculin to vinculin
Bind anti-vinculin to vinculin
Remove blocking buffer.
Bind anti-vinculin to vinculin
Bind anti-vinculin to vinculin
Wash twice with washing buffer.
Bind anti-vinculin to vinculin
Bind anti-vinculin to vinculin
Add 250 µL of Anti-Vinculin and blocking buffer mixture.
Bind anti-vinculin to vinculin
Bind anti-vinculin to vinculin
Incubate for 00:20:00 .
Stain actin filaments and focal adhesion sites
Stain actin filaments and focal adhesion sites
Remove anti-vinculin blocking buffer mixture.
Stain actin filaments and focal adhesion sites
Stain actin filaments and focal adhesion sites
Wash twice.
Add 100 µL FITC:TRITC solution, cover in foil.
Incubate for 00:30:00 .
Stain nuclei
Stain nuclei
Remove stains.
Add 100 µL of DAPI solution, cover in foil.
Incubate for 00:05:00
Remove DAPI solution.
Add 100 µL PBS.
Image
Image
Image your cells using UV, Blue, and Green excitation.