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

In This Article

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.
1. Remove the media from each well of the plate containing organoids by tilting the plate at a 45° angle.
2. Digest the matrix gel by incubating it with ....

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Results

Primary organoids: microscopy images, protein expressions, and coverslip setup.
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.......