Gtvision detection system mo rb

For each patient, one tumor tissue block with both tumor and invasive margins was chosen and made into 4 μm-thick paraffin-embedded tissue sections. Two tissue sections were dewaxed with xylene and rehydrated with decreasing concentrations of gradient alcohol. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide for 15 min. Antigen retrieval was performed in the steamer for 20 min with citrate antigen retrieval solution (pH 6.0). Nonspecific antigens were blocked with 10% normal goat serum for 1 hour at 37°C. Then, the tissue sections were incubated with anti-CD3 antibody (ab5960, goat multiclonal, 1 : 200, Abcam, Cambridge, UK) and anti-CD8 antibody (#70306, mouse monoclonal, 1 : 500, Cell Signalling Technology, Danvers, MA, USA) in the wet box overnight at 4°C. Subsequently, the secondary antibody (GK5005, GTVision™ Detection System/Mo&Rb, Gene Tech, Shanghai, China) was incubated for 30 min at 37°C, and diaminobenzidine (DAB) was used for chromogen reaction. After all of the above steps, all sections were counterstained with hematoxylin-eosin solution and dehydrated with increasing xylene and alcohol gradient solutions.

Pancreas tissues were separated, fixed, and embedded in paraffin [21 (link)]. Serial sections were deparaffinized with dimethylbenzene, and antigen retrieval was conducted using citrate buffer solution. The slides were incubated with 3% hydrogen peroxide for 15 min and then blocked with 10% goat serum for 1 hour at room temperature. The slides were incubated with primary antibodies targeting Ngn3 (AVIVA SYSTEMS BIOLOGY, OAAB15617), cleaved Notch1 (Abcam, ab8925), insulin (Abcam, ab7842), NKX6-1 (Cell Signaling Technology, #54551), glucagon (Sigma, G2654), PDX-1 (Abcam, ab47267), Ki67 (Abcam, ab15580), and caspase12 (Abcam, ab62484) at 4°C in a refrigerator overnight. For immunohistochemistry, the GTVision™+ Detection System/Mo&Rb (Gene Tech, GK6005) was used to examine the signal based on the guidelines. For immunofluorescence staining, the signal was detected using FITC-labelled IgG fluorescence.

The pulmonary tissues were prepared by being submerged in formaldehyde solution (10%) and embedded in paraffin. The paraffin blocks were cut at 5 μm using microtome. The sections stained with hematoxylin and eosin (H&E) staining and Masson's trichrome staining following the manufacturers’ instructions. Immunohistochemistry was used to investigate the expression of α‐SMA and FOXO3a using GTVisionTM + Detection System/Mo&Rb (Gene Tech, Shanghai, China) according to instruction book. After water‐bath heating for antigen retrieval in 0.01 mol/L citrate buffer (pH 6.0), 30% H2O2 was added to the sections to inactivate the endogenous enzymes at room temperature for 10 minutes. Then, the slides were blocked with 5% BSA blocking solution at room temperature for 15 minutes and incubated with primary anti‐α‐SMA (1:400) and anti‐FOXO3a (1:500) antibodies overnight at 4°C. Negative controls were performed by omitting the primary antibody. Subsequently, the sections were incubated with general horseradish peroxidase‐labelled secondary antibody at working fluid for 30 minutes at 37°C, and developed with diaminobenzidine (DAB) for colour reaction. The nuclear staining with hematoxylin and then using a light microscope (Leica DM3000, Leica Company, Germany).