Immunostaining of Cells Encapsulated in a 3D Hydrogel System

Abstract

Source: LeSavage, B. L., et al. Production of Elastin-like Protein Hydrogels for Encapsulation and Immunostaining of Cells in 3D. J. Vis. Exp. (2018).

This video demonstrates a technique for immunostaining cells encapsulated in a 3D hydrogel. The hydrogel culture containing neural progenitor cells is fixed, permeabilized, and treated with a blocking solution to prevent non-specific immunostaining. A primary antibody cocktail is added to bind specific proteins, followed by fluorescently labeled secondary antibodies and a nuclear stain. The sample is then mounted on a slide and observed under a confocal microscope.

Protocol

1. Cell Encapsulation in 3D Elastin-like Protein (ELP) Hydrogels Preparation of silicone molds Use a biopsy punch with the desired diameter to create holes in a 0.5 mm thick silicone sheet and cut out a square around each hole. Repeat for the number of molds desired. ​NOTE: The diameter and thickness of the molds can be adjusted for the particular application and cell culture conditions. In practice, for cell cultures of 50 x 10…

Materials

Cell Encapsulation in 3D ELP Hydrogels
0.22 μm syringe filters	Millipore	SLGV004SL
0.5 mm thick silicone sheet	Electron Microscopy Science	70338-05
24-well tissue culture plates	Corning	353047
Disposable Biopsy Punch (2 mm)	Integra Miltex	33-31
Disposable Biopsy Punch (4 mm)	Integra Miltex	33-34
Disposable Biopsy Punch (5 mm)	Integra Miltex	33-35
Dulbecco’s phosphate buffered saline (DPBS)	Corning	21-031-CM
No. 1 12 mm glass coverslips	Thermo Fisher Scientific	12-545-80
Tetrakis(hydroxymethyl)phosphonium chloride (THPC)	Sigma-Aldrich	404861-100ML
0.5% Tryspin/EDTA	Thermo Fisher	15400054
Immunocytochemistry of Cells in 3D ELP Hydrogels
16% (w/v) Paraformaldehyde (PFA)	Electron Microscopy Sciences	15701
Bovine Serum Albumin (BSA)	Roche	3116956001
DAPI (4',6-Diamidino-2-Phenylindole, Dihydrochloride)	Molecular Probes	D1306
Donkey Serum	Lampire Biological Labs	7332100
Goat anti-mouse Secondary Antibody (AF488)	Molecular Probes	A-11017
Goat anti-rabbit Secondary Antibody (AF546)	Molecular Probes	A-11071
Goat Serum	Gibco	16210-072
Mouse Nestin Primary Antibody	BD Pharmingen	556309
Mouse Sox2 Primary Antibody	Cell Signaling Technology	23064S
Nail Polish	Electron Microscopy Sciences	72180
Triton X-100	Sigma-Aldrich	X100-100ML
Vectashield Hardset Mounting Medium	Vector Labs	H-1400