Ab15160

Thick 100 μm cryostat sections were rehydrated in PBS. Sections and whole mount pancreata were blocked overnight in PBS, 2% donkey serum and 0.5% Triton-100X. The samples were incubated in primary antibodies (Chromogranin A, 1:180 from Abcam, ab15160; insulin, 1:100, from Abcam, ab7842; glucagon 1:200, from Abcam, ab10988); and Dolichos biflorus agglutinin (DBA) biotinylated (1:270, from Vectorlabs, B-1035) for 3 days at 4 °C. Secondary antibodies were applied (from Thermo Fisher Scientific) and AF647-Streptavidin (1:800, from Thermo Fisher Scientific, S21374), BV510-Streptavidin (1:600, from Biolegend, 405233) for 2 days at 4°C. All tissues were cleared with RapiClear 1.52 (from SunJin Lab, RC152001).
Thin sections were incubated in 2% Serum, 0.1% Triton-100X in PBS for 30 min, subsequently incubated in primary antibody in 0.01% Triton-100X in PBS overnight (insulin 1:100, from Abcam, ab7842, glucagon 1:200 from Abcam, ab10988, cleaved caspase 3, 1:400, from Cell Signalling, 9664S), washed 3 times for 10 min the next day, and incubated with secondary antibody in 0.01% Triton-100X for 3 h, washed and mounted.

Paraffin-embedded specimens from patients with primary and relapsed castration-resistant PCa that had been collected between 1990 and 2010 at Mackay Memorial Hospital were included in this study. Ethics was approved by the Mackay Memorial Hospital Institute Review Board. Informed consent was written. Tissue sections were deparaffinized in xylene, rehydrated through graded ethanol (100, 90, 80, 70, 50%), subjected to antigen retrieval by microwaving in 10 mM citrate buffer (pH 6.0) for 10 min, blocked for endogenous peroxidase activity with 3% H₂O₂ for 10 min at room temperature, and then blocked with antibody diluent containing background-reducing components (DAKO, S302281). Finally, the slides were stained overnight at 4°C with beclin1 (GeneTex, GTX61619), LC3 (Novus Biologicals, NB110-57179), REST (BETHYL, IHC-00141), or CgA (abcam, ab15160) specific antibodies and visualized following standard protocol using 3-amino-9-ethylcarbazole (AEC) as chromogen (DAKO, K346430) in the presence of hematoxylin counterstaining. The histology and staining was evaluated by a pathologist in a blind fashion using a four-tier scale representing negative (0), weak (1), intermediate (2), and strong (3) signals.

Immunofluorescence was performed as previously described (Li et al. 2018 (link)). Briefly, organoids were fixed in 4% paraformaldehyde for 1 h at room temperature. Organoids were washed by PBS for 3 times and permeabilized by 0.5% Triton X-100 for 1 h at room temperature. Then, samples were blocked with PBT solution (3% BSA and 0.01% Triton X-100 in PBS) for 2 h at room temperature and incubated with primary antibodies overnight at 4 °C. The fluorescein-labeled secondary antibodies (Life Technologies, 1:300) and 4′, 6-diamidino-2-phenylindole (DAPI) were added for 1 h at room temperature next day. The following antibodies were used for immunofluorescence: rabbit anti-Fabp1 (Abcam, ab171739, 1:300); mouse anti-E-cadherin (B&D, 610182, 1:300); rabbit anti-Muc2 (Santa Cruze, sc-15334, 1:300); rabbit anti-ChgA (Abcam, ab15160, 1:300). The images were acquired from Olympus FV3000 Laser Scanning Microscope.

Immunostaining for lysosome, Sucrase-Isomaltase, Ki-67, mMECA-32, and ChromograninA was performed on frozen sections using rabbit anti-lysozyme (A0099, DAKO, 1:1000), mouse anti-sucrase-isomaltase (sc-393470, clone: C-8, Santa Cruz, 1:50), rabbit anti-ki-67 (ab16667, clone: SP6, abcam, 1:500), rat anti-MECA-32 (NB100-77668, clone: MECA-32, NOVUS Biologicals, 1:50), and rabbit anti-ChoromograninA (ab15160, Abcam, 1:400) antibodies, respectively. Immunostaining for GFP to detect Lgr5 expression was performed on frozen sections or paraffin-embedded sections using rabbit anti-GFP (2956, clone: D5.1, Cell Signaling, 1:100) antibody. For DAB staining, slides were then incubated with amino acid polymer conjugated with peroxidase and Fab’ (Histofine simple stain rabbit MAX-PO (R), 414341, Nichirei Bioscience Inc.). Slides were visualized using metal-enhanced 3,3′- diaminobenzidine (DAB) and counterstained with hematoxylin. Images were acquired using an OLYMPUS BX41 microscope (OLYMPUS Corporation). When co-staining of Lys and GFP or co-staining with rainbow colors were performed, Alexa488-, Alexa594-, and Alexa750-conjugated secondary antibodies (1:200, Invitrogen) were used, followed by nuclei-staining using Hoechst33342 (Thermo Fisher Scientific, H3570). Fluorescent images were taken with an OLYMPUS BX63 microscope (Olympus Corporation, Tokyo, Japan).