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