Ez retriever system

Whole pancreata were removed from WT and β14-3-3ζ–KO mice, weighed, fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned to 6 μm thickness. A minimum of 3 sections, 72 μm apart, were used in all studies. Antigen retrieval at 95°C was performed using EZ-Retriever System (Biogenex) with 10 mM sodium citrate buffer. β Cell proliferation was assessed, as described above. The In Situ Cell Death Detection Kit (TUNEL; Roche Applied Sciences) was used to measure β cell apoptosis (Roche; ref. 15 (link)). To measure β cell mass in pancreatic sections, IHC was performed with an insulin antibody (1:200 dilution; Ab 3014, Cell Signaling Technology) and the SignalStain DAB Substrate Kit (Cell Signaling Technology). Hematoxylin was used for counterstaining. Slides were monitored using a high-resolution scanner (Aperio ImageScope 12.3.3) to assess the areas of insulin⁺ β cells and the whole pancreas, followed by calculating β cell mass (total β cell area/total pancreas weight).

To simultaneously detect GLI1 and cell phenotype markers a mfIHC was performed using tyramide signal amplification approach as previously reported [19 (link), 53 (link)]. Briefly, human BM tissue sections dried at 60 °C were deparaffinized and rehydrated in xylene-ethanol gradient, fixed with 3% hydrogen peroxide/methanol solution, and put through five or six rounds of staining. Each round consisted of antigen retrieval at 95 °C using Citra Plus in an EZ-Retriever system (BioGenex, Fremont, CA, USA), block with 3% bovine serum albumin, incubation with primary antibody (Supplementary Table S3, human panel A), and detection using SuperPicture broad-spectrum horseradish peroxidase polymer antibodies (Invitrogen) and an Opal fluorophore (Supplementary Table S5). DAPI (4′,6-diamidino-2-phenylindole; Invitrogen) was used as a nuclear counterstain. Xenograft mouse and human panel B tissue sections were stained by adapting the above protocol on a NanoVIP automated staining system (BioGenex). Deparaffination and antigen retrieval were performed using X-DeWax and EZ-AR1 or EZ-AR2 solutions (BioGenex). Blocking and detection of the primary antibodies were achieved using 2.5% goat serum solution and ImmPRESS species-specific horseradish peroxidase polymer antibodies (Vector Biolabs, Malvern, PA, USA), respectively.