Citrate unmasking solution

Patient tissues and PDXs were embedded in paraffin. After this, we generated 4-μm-thick paraffin sections. Tissue morphology was determined by hematoxylin and eosin (H&E) staining. For immunohistochemistry (IHC) assays, paraffin sections were dewaxed and rehydrated through a graded ethanol series. After being treated with heat-mediated antigen retrieval using Citrate Unmasking Solution (Cell Signaling Technology), the sections were blocked with goat serum at room temperature for 1 h. Primary antibodies (Additional file 2: Table S4) diluted in 3% BSA were incubated at 4 °C overnight. We used DAB (Cell Signaling Technology) to perform the color reaction. All H&E and IHC images were obtained using an OLYMPUS microscope BX51 and a DP 71 camera (OLYMPUS). The positive grade was determined by the immuno-reactive score (IRS), which was according to the staining intensity (negative = 0, weak = 1, moderate = 2, strong = 3) and the percentage of positive tumor cells (0%=0, 1–10%=1, 11–50%=2, 51–80%=3, 81–100%=4). IRS ranging from 0 to 12 was calculated from the values of the staining intensity multiplied by the percentage of positive cells [31 (link)].

Briefly, 5 µm thick paraffin sections of pancreatic tissues were collected onto positively charged slides prior to staining with hematoxylin and eosin (H&E) for microscopic evaluation or immunofluorescence assay (IFA), as previously described [14 (link)]. For IFA, slides were rehydrated using previously outlined methods of toluene and ethanol changes [14 (link)] followed by antigen retrieval using Citrate Unmasking Solution (Cell Signal, Danvers, MA, USA). Slides were blocked with 10% goat serum for 1 h followed by overnight incubation with a rabbit polyclonal anti-insulin antibody (4590, Cell Signaling Technology, Danvers, MA, USA). Anti-rabbit Alexa Fluor 555 was used as the secondary antibody, as well as DAPI for a nuclear counterstain (Cell Signaling Technology, Danvers, MA, USA). All slides were assigned a quantitative histologic score based on relative total number of cells and fluorescent intensity by a board-certified veterinary pathologist blinded to study groups to ensure scientific rigor and reproducibility.