Fluorescence Staining of Neural Crest Cells Cultured on Hydrogels of Varying Stiffness

Abstract

Source:  Le, T. P., et al., An Optimized O9-1/Hydrogel System for Studying Mechanical Signals in Neural Crest Cells. J. Vis. Exp. (2021)

In this video, we describe the procedure to perform fluorescence staining using fluorescence microscopy to demonstrate phalloidin staining of neural crest cells cultured on hydrogels of varying stiffness to visualize and analyze cytoskeletal organization.

Protocol

1. Molecular analysis of stiffness via immunofluorescence staining

1. Use tweezers to transport the coverslip to a new plate to minimize false signals from cells grown directly onto the plate. Wash the cells with 500 µL of sterile PBS three times to remove dead cells and any remaining culture medium.
2. Fix the cells using undisturbed 500 µL of 4% paraformaldehyde (PFA) for 10 min at room temperature. Then, rewash the cells three times using 500 µL of PBS/well for 2 min each.
   NOTE: Store at 4 °C for a procedural stop.
3. Treat the cells with 500 µL of 0.1% Triton X-100 for 15 min ....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
2 mm #1 Corning 0211 Glass Coverslip	Chemglass Life Sciences	CLS-1763-012
25 mm #1 Corning 0211 Glass Coverslip	Chemglass Life Sciences	CLS-1763-025
4-well cell culture plate	Thermo Scientific	179830
4% Paraformaldehyde	Sigma Aldrich	J61899-AP
40% Acrylamide	Sigma Aldrich	A4058
Alexa Fluor 488 Phalloidin	Thermo Fisher	A12379
Collagen type I (100mg)	Corning	354236
DAPI (4',6-Diamidino-2-Phenylindole, Dihydrochloride)	Thermo Fisher	D1306
Donkey serum	Sigma Aldrich	D9663
Sterile 1x PBS	Hyclone	SH30256.02
Fluorescence microscope	Leica