Immunofluorescent Staining of Spheroids: A Technique to Analyze Spheroids for Expression of Intra- and Extracellular Antigen Expression

Published: April 30, 2023

Abstract

Source: Cavaco, A. C. M. et al. A 3D Spheroid Model as a More Physiological System for Cancer-Associated Fibroblasts Differentiation and Invasion In Vitro Studies. J. Vis. Exp. (2019).

This video describes the technique of immunofluorescent staining of spheroids to understand the differentiation of the fibroblasts or CAFs. This protocol helps detect expression of intra- and extracellular antigens, including deposition of ECM proteins within the spheroids.

Protocol

1. Immunofluorescent staining of spheroids Collect the spheroids after about 24 h into one 1.5 mL reaction tube per experimental condition or per protein to be analyzed. For example, place the spheroids of iNFs stimulated with TGF-β1 in a tube different from the control iNFs, which were not treated. To avoid rupture of spheroids due to too strong shear forces, cut the end of the pipette tip to enlarge the orifice. Include at least 5-10 spheroids in one reaction tube, to allow replicates and take into acc…

Representative Results

Figure 1. Sequential steps for quantification of the immunofluorescent staining of a protein of interest in spheroids, using ImageJ. Please click here to view a larger version of this figure.

開示

The authors have nothing to disclose.

Materials

4',6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI)   SIGMA-ALDRICH D9542-10
6F12 anti-Laminin β3 subunit Mouse (monoclonal)  Homemade
Albumin Fraction V – BSA   AppliChem A1391
Alexa fluor 488 Goat (polyclonal) anti-Mouse   Invitrogen A11029
Alexa fluor 488 Goat (polyclonal) Rabbit   Invitrogen A11034
Anti-laminin γ2 subunit Mouse (monoclonal)  Santa Cruz  sc-28330
Bench centrifuge   Fisher Scientific 50-589-620  Sprout
BM2 anti-laminin α3 subunit Mouse (monoclonal)  Kindly provided by Prof. Patricia Rousselle, CNRS, Lyon
Centrifuge tubes 50 mL  Corning 430290
Confocal microscope  Zeiss
DMEM (High glucose 4.5 g/L)  Lonza  BE12-604F
Human Primary Pancreatic Fibroblasts  PELOBiotech  PB-H-6201
Penicillin/streptomycin  Gibco  15140-122
Microscope Slides  Thermo Scientific  J1800AMNZ
Paraformaldehyde   Riedel-de Haën 16005
TritonX-100 SIGMA-ALDRICH   X100RS

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記事を引用
Immunofluorescent Staining of Spheroids: A Technique to Analyze Spheroids for Expression of Intra- and Extracellular Antigen Expression. J. Vis. Exp. (Pending Publication), e20312, doi: (2023).

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