Ab5450

Paraffin sections were de-waxed and rehydrated. Antigen retrieval was carried out by microwaving the slides in 0.01 M sodium citric acid buffer (pH 6.0) for 30 min. Sections were then immersed for 1 hour in blocking buffer (3% BSA, 0.2% Triton X-100 in PBS), then incubated in primary antibody (in blocking buffer) at 4° C overnight, followed by incubation with secondary antibody at 4° C for 1 hour. Slides were mounted with antifade reagent with or without DAPI (Life Technologies), and then scanned with a high-resolution MIRAX MIDI system (Carl Zeiss) equipped with both bright field and fluorescence illumination. Images were analyzed by the MIRAX Viewer software.
Polyclonal rabbit anti-Scgb1a1 antibody (US Biological, C5828) was used at 1:200 dilution. Goat anti-pro-SPC (Santa Cruz Biotechnology, sc-7706), rabbit anti-Cyp2f2 (Santa Cruz Biotechnology, sc-67283), goat anti-PDPN (R&D Systems, AF3244), monoclonal mouse anti-p63 4A4 (Santa Cruz Biotechnology, sc-8431), rabbit anti-GFP (Abcam, ab290), goat anti-GFP(Abcam, ab5450), and mouse anti-GFP (Abcam, ab1218) were used at 1:50 dilution. Secondary antibodies (including donkey anti-rabbit, anti-goat, or anti-mouse) each with different Alexa Fluor conjugations were all purchased from Life Technologies, and used at 1:200 dilution.

Organoids were retrieved from Matrigel using Cell Recovery Solution (Corning) and fixed in 4% paraformaldehyde in PBS (Alfa Aesar) for 30 min at room temperature. 2D cultures on glass coverslips were fixed in 4% paraformaldehyde in PBS for 20 min. Immunostaining was performed as previously described [17] . Rabbit polyclonal antibodies against proglucagon (Santa Cruz, sc-13091) were used at 1:200 and goat polyclonal antibodies against GFP (Abcam, ab5450) at 1:1000 to detect YFP and GCaMP3. Secondary antibodies conjugated to Alexa-Fluor 488 and 555 (Invitrogen) were used at 1:1000 and Hoescht (Sigma) nuclear stain at 1:3000.

The organoids were then formalin fixed and paraffin embedded. Sections were deparaffinized with xylene, treated with antigen retrieval solution (1 g NaOH, 2.1 g citric acid in 1 L of H₂O) for 20 min in steam, cooled to room temperature, permeabilized with PBS-0.2% Triton for 15 min, and blocked with 10% donkey serum in PBS. Sections were stained with monoclonal rabbit antithyroid transcription factor 1 (TTF1, ab76013; Abcam, Cambridge, MA) or rabbit anti-CC10 (ab40873, Abcam, Cambridge, MA, USA) at 1:500 followed by Alexa 594-conjugated donkey antirabbit secondary antibody (ab150076, Abcam, Cambridge, MA, USA) at 1:500, goat anti-GFP (ab5450; Abcam, Cambridge, MA, USA) at 1:500 followed by Alexa 488-conjugated donkey anti-goat secondary antibody (ab150129, Abcam, Cambridge, MA, USA) at 1:500. Images were taken at 10×, 20×, and 40× with an Olympus FV1000 confocal microscope. Image analysis were performed using ImageJ software.

Sorted cells were collected in 200 µl of 1× PBS and seeded in a Nunc glass-bottom dish (Thermo Fisher Scientific, #150680) previously treated with 200 µl of 20 µg ml⁻¹ fibronectin (Sigma-Aldrich, #F1141-2MG) overnight at 4°C. Cells were incubated for 3 h at 23°C, then 1× PBS was substituted with 200 µl growth medium and grown overnight at 23°C.
Cells were fixed for 5 min at room temperature with 4% formaldehyde in 1× PBS and washed once with 200 µl 1× PBS. Cells were then blocked for 30 min at room temperature in blocking solution [1% bovine serum albumin (Sigma-Aldrich, #A3294-10G), 0.1% Triton-X100 (Sigma-Aldrich, X100) in 1× PBS] and incubated for 1.5 h at room temperature with 1:100 anti-green fluorescent protein (GFP) primary antibody (Abcam, ab5450, Lot GR277059-1) in blocking solution [Venus is an improved version of GFP (Nagai et al., 2002 (link))]. Cells were washed twice for 10 min in blocking solution and incubated for 1.5 h in the dark at room temperature with 1:1000 Alexa Fluor 568 goat anti-rat IgG (Life Technologies, A11077, Lot 1512105) in the blocking solution. After three washes of 10 min in 1× PBS, the preparation was overlaid with fluorescence mounting media (DAKO/Agilent Technologies, #S3023), covered with a coverslip and sealed with nail polish.

LNCaP, PC3 and MCF7 cells were grown for 48 hours and incubated with 25nM GFP-SCP for 5 hours at 37°C. After incubation cells were fixed with 4% paraformaldehyde, washed twice with PBS, permeabilized and stained with goat anti-GFP antibody (1:1000, ab5450, Abcam, Cambridge, MA, USA ), followed by incubation with DyLight 488-conjugated anti-goat secondary antibody (1:300, Jackson ImmunoResearch Laboratories, West Grove, PA, USA). 4, 6-diamidino-2-phenylindole (DAPI) was used to stain DNA. Stained samples were observed with a confocal microscope, FLUOVIEW FV-1000 (Olympus, Tokyo, Japan).