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

Samenvatting

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 at room temperature. Then, wash the cells three times with 500 µL of PBS/well.
4. Block the cells with 250 µL of 10% donkey serum (diluted in PBS and 0.1% Tween 20) per well for 30 min at room temperature.
5. Incubate the cells with phalloidin used for F-actin staining at a dilution of 1:400 in 250 µL of 10% donkey serum for 30 min at room temperature. Then, wash the cells three times with PBS for 5 min each.
   NOTE: 568 nm Phalloidin can be co-incubated with 488 nm or 647 nm secondary antibodies or on its own.
6. Incubate the cells with 4′,6-diamidino-2-phenylindole (DAPI, 1:1000 dilution) in 250 µL of PBS for 10 min followed by one last wash of PBS for 2 min.
7. Add 3-4 drops of mounting medium to each well. Store the samples at 4 °C to set for at least 2 h before imaging to ensure the mounting medium has been set properly.
8. With a fluorescence microscope, capture images of at least 3 random frames per hydrogel sample, producing individual and merged channels.

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
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