Whole-mount Immunofluorescence Imaging: A Fixing and Staining Technique to Evaluate Intact Organoids

Published: April 30, 2023

Abstract

Source: Crowell, P. D. et al. Evaluating the Differentiation Capacity of Mouse Prostate Epithelial Cells Using Organoid Culture. J. Vis. Exp. (2019)

This video describes the technique of immunofluorescent staining of intact prostate cancer organoids and their imaging by whole-mount confocal microscopy. This technique aims to evaluate the ex vivo differentiation capacity of basal and luminal prostate epithelial cells.

Protocol

1. Fixing and Staining Prostate Organoids for Immunohistochemical Analysis by Whole-mount Confocal Microscopy Collecting prostate organoids from 24-well plates — TIMING: 45-60 min NOTE: When collecting prostate organoids to process for confocal microscopy, it is critical to handle them with care in order to maintain their structure. The collection protocol below is designed to reduce disruption of the organoid structure during isolation. Rem…

Representative Results

Figure 1: Analysis of lineage marker expression in prostate organoids by Western blot and whole-mount confocal microscopy. (A) Representative phase contrast images of basal-derived (top), and luminal-derived (bottom) organoids after 7 days of culture. Scale bar = 100 µm. (B) Western blot analysis of basal-derived (Bas) and luminal-derived (Lum) organoid…

Divulgaciones

The authors have nothing to disclose.

Materials

16% Paraformaldehyde   Thermo Fisher Scientific 50-980-487
4’,6-diamidino-2-phenylindole (DAPI)  Thermo Fisher Scientific  D1306
Advanced DMEM/F-12   Thermo Fisher Scientific 12634010
Dispase II, Powder   Thermo Fisher Scientific 17-105-041
Fetal Bovine Serum (FBS)   Sigma F8667
Goat anti-mouse IgG-Alexa Fluor 488   Invitrogen A28175
Goat anti-rabbit IgG-Alexa Fluor 594   Invitrogen A11012
Mouse anti-cytokeratin 8   BioLegend 904804
Penicillin-Streptomycin (10,000 U/ mL)   Thermo Fisher Scientific 15-140-122
Rabbit anti-p63  BioLegend 619002
RPMI 1640 Medium, HEPES (cs of 10)  Thermo Fisher Scientific 22400105
Sucrose   Sigma S0389-500G
Triton X-100  Sigma  X100-5ML

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Citar este artículo
Whole-mount Immunofluorescence Imaging: A Fixing and Staining Technique to Evaluate Intact Organoids. J. Vis. Exp. (Pending Publication), e20391, doi: (2023).

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