Antigen retrieval citra plus

Pancreatic tissues were fixed in 10% neutral-buffered formalin (FisherBrand) and then embedded in paraffin and sectioned into slides. For IHC, fresh-cut paraffin slides were rehydrated using two series of xylene, two series of 100% ethanol, and then two series of 95% ethanol. Water was used to wash all residues from previous washes. Antigen retrieval was performed using Antigen Retrieval CITRA Plus (BioGenex) and microwaved for a total of 8 min. Upon cool-down, tissue was blocked using 1% BSA in PBS for 30 min, and then primary antibodies in Table S1 were used at their corresponding dilutions. Biotinylated secondary antibodies were used in 1:300 dilution. Following the secondary antibody incubation, the tissue was incubated for 30 min with the ABC reagent from VECTASTAIN Elite ABC kit, peroxidase (Vector Laboratories). Then it was developed using DAB (Vector). For IF, Alexa fluor secondary antibodies (1:300, Invitrogen) were used. Prolong Diamond Antifade Mountant with DAPI (Invitrogen) was used for nuclei staining. The Tyramide SuperBoost Kit (Invitrogen) was used in IF when primary antibodies raised in the same species were used. Images were taken with an Olympus BX-53 microscope, Olympus DP80 digital camera, and Olympus cellSens standard software. Some IF images were acquired using the Leica Stellaris 5 confocal microscope (Leica Microsystems).

Immunostaining was performed according to prior reports^{[1 (link)]}. Briefly, heart cryosections were equilibrated with antigen retrieval buffer in epitope retrieval buffer (IHC World) or 1× citrate buffer (Antigen Retrieval Citra Plus, Biogenex). Samples were permeabilized and blocked with 0.3% Triton X-100 and 10% serum from the host animal of secondary antibodies in PBS for 1 h at room temperature. Then, the samples were incubated overnight at 4 0C with primary antibodies. After three washes in PBS, samples were incubated at room temperature for 1h with the corresponding fluorescence secondary antibodies conjugated to Alexa Fluor 488 or 555 (Invitrogen) at 1:400. The slides were mounted in Vectashield Antifade Mounting Medium (Vector Laboratories). Slides were viewed under Nikon fluorescence or Zeiss LSM 510 confocal microscopes. Primary antibodies: pH3 Ser10 (EMD Millipore, 06–570; 1:100); troponin T (Thermo Scientific, MS-295-P1; 1:200); 8-oxoG (Abcam ab64548, 1:25). DAPI was used for nuclear staining. Images were obtained on a Nikon Eclipse Ni or Nikon A1 laser scanning confocal microscopes.