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

Overview

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

1. 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 500 µL of dispase-containing media (Table 1) for 30 min in a 37 °C 5% CO₂ incubator.
   3. Collect digested organoid suspension in a microcentrifuge tube and pellet the organoids by centrifugation at 800 x g for 3 min at RT. Remove the supernatant.
2. Whole-mount immunofluorescent staining of prostate organoids — TIMING: 3-4 days (1-5 h/day)
   1. Add 500 µL of 4% paraformaldehyde in PBS and incubate for 2 h at RT with gentle shaking.
   2. Pellet the organoids by centrifugation at 800 x g for 3 min at RT, remove the supernatant, and wash the pellet with 1 mL of PBS for 15 min with gentle shaking.
   3. Wash the pellet as in step 1.2.2 for an additional two times.
   4. Pellet the organoids by centrifugation at 800 x g for 3 min at RT and remove the supernatant. Add 1 µg/mL DAPI in blocking solution (Table 1). Incubate for 2 h at RT or alternatively overnight at 4 °C with gentle shaking.
   5. Pellet the organoids by centrifugation at 800 x g for 3 min at RT and remove the supernatant. Add primary antibody (rabbit anti-p63, mouse anti-cytokeratin 8) in blocking solution and incubate overnight at 4 °C with gentle shaking.
   6. Pellet the organoids by centrifugation at 800 x g for 3 min at RT and remove the supernatant. Wash the pellet with 1 mL of PBS for 15 min with gentle shaking.
   7. Wash the pellet as in step 1.2.6 for an additional two times.
   8. Pellet the organoids by centrifugation at 800 x g for 3 min at RT and remove the supernatant. Add secondary antibody (goat anti-rabbit IgG-Alexa Fluor 594, goat anti-mouse IgG-Alexa Fluor 488) in blocking solution and incubate overnight at 4 °C with gentle shaking.
   9. Pellet the organoids by centrifugation at 800 x g for 3 min at RT, remove the supernatant, and wash the pellet with 1 mL of PBS for 15 min with gentle shaking.
   10. Wash the pellet as in step 1.2.9 for an additional two times.

2. Tissue Clearing and Mounting of the Stained Prostate Organoids for Whole-mount Confocal Microscopy — TIMING: 7 H

1. Pellet the organoids by centrifugation at 800 x g for 3 min at RT and remove the supernatant.
2. Add 1 mL of 30% sucrose in PBS with 1% Triton X-100 and incubate for 2 h at RT with gentle shaking.
3. Pellet the organoids by centrifugation at 800 x g for 3 min at RT and remove the supernatant.
4. Add 1 mL of 45% sucrose in PBS with 1% Triton X-100 and incubate for 2 h at RT with gentle shaking.
5. Pellet the organoids by centrifugation at 800 x g for 3 min at RT and remove the supernatant.
6. Add 1 mL of 60% sucrose in PBS with 1% Triton X-100 and incubate for 2 h at RT with gentle shaking.
7. Pellet the organoids by centrifugation at 800 x g for 3 min at RT and remove 95% of the supernatant.
   NOTE: The pellet becomes looser as the concentration of sucrose becomes higher. Observing the DAPI-stained organoids under the UV light to confirm that they were not lost during removal of the supernatant is recommended.
8. Transfer a 10-20 µL droplet of the remaining suspension to a chambered coverslip and proceed to confocal microscopy.
   NOTE: Coverslip fragments can be placed on either side of the droplet to be used as spacers (Figure 1C). These prevent organoids from collapsing when a coverslip is placed over the droplet.

Table 1: Instructions for the preparation of key solutions

Component	Concentration
B-27	1x (dilute from 50x concentrate)
GlutaMAX	1x (dilute from 100x concentrate)
N-acetyl-L-cysteine	1.25 mM
Normocin	50 µg/mL
Recombinant Human EGF, Animal-Free	50 ng/mL
Recombinant Human Noggin	100 ng/mL
R-spondin 1-conditioned media	10% conditioned media
A83-01	200 nM
DHT	1 nM
Y-27632 dihydrochloride (ROCK inhibitor)	10 µM
Advanced DMEM/F-12	Base media
R-spondin 1-conditioned media is generated as described in Drost, et al. After addition of all components, filter sterilize mouse organoid media using 0.22 µm filter. ROCK inhibitor is only added during establishment of culture and passaging of organoids.

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) organoids after 5 days of culture. Staining for the basal marker, cytokeratin 5 (K5), and the luminal marker, cytokeratin 8 (K8), and a loading control, histone H3 (HH3). (C) Schematic illustrating chambered coverslip with spacers. (D) Representative differential interference contrast (DIC) and immunofluorescent images of basal-derived (top) and luminal-derived (bottom) organoids after 7 days of culture. Staining for p63 (red), K8 (green) and DAPI (blue) individually and merged. Scale bars = 100 µm.

Materials

Name	Company	Catalog Number	Comments
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