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.
Divulgaciones
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 |
Citar este artículo
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).