Rabbit anti insulin antibody

A portion of the pancreatic tail was fixed in 10% buffered formalin, embedded in paraffin, sectioned and stained with Hematoxylin and Eosin (H&E). For fluorescent microscopy imaging of insulin and glucagon, paraffin sections (2 μm) were put onto microscope slides, deparaffinized in xylene, and hydrated through graded ethanol to distilled water. Tissue sections were permeabilized in PBS with 0.1% Triton X-100 for 10 min. Blocking was performed using PBS with 5% normal goat serum for 1 h at room temperature, followed by incubation with rabbit anti-insulin antibody (Cell Signaling Technology, Danvers, MA, USA) and mouse anti-glucagon antibody (Abcam, Cambridge, UK) diluted 1:100 at 4°C overnight. Three consecutive washes with PBS for 5 min each were followed by sequential incubation with Alexa Fluor 595 and 488 goat anti-rabbit and anti-mouse IgG secondary antibodies (1:200) at room temperature for 1 h. The slides were washed three times with PBS and mounted using an anti-fade mounting medium containing 4',6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Burlingame, CA, USA). Images were captured under a fluorescence microscope (Carl Zeiss AG; Oberkochen, Germany). Islet area was determined from at least five different islets per pancreas section stained with H&E using Image J software (National Institute of Health, NIH Version v1.32j).

Pancreatic tissues were harvested and fixed with 4% PFA overnight at 4 °C and tissues were processed and embedded in paraffin blocks as described previously (80 ). Tissue sections were stained with rabbit anti-insulin antibody (Cell Signaling) and counterstained with DAB peroxidase anti-rabbit IgG (Vector Lab). Images were acquired using a Zeiss slide scanner (Zeiss, Germany), and insulitis scoring was performed on 5 slides, each 30 μm apart, from 9 mice/group as previously described (80 ). Immunofluorescence studies were performed according to the protocol described previously (46 (link)) using the following antibodies: guinea pig anti-insulin (Dako), mouse anti-PD-L1 (Proteintech), and rabbit anti-CXCL10 (Invitrogen). Signals were detected by counter staining with the following antibodies: goat anti-guinea pig (1:400; Invitrogen), goat anti-rabbit (1:200; Invitrogen), and goat anti-mouse (1:200; Invitrogen). All sections were counterstained with DAPI to identify nuclei. Images were obtained with a Zeiss LSM 800 confocal microscopy (Carl Zeiss, Germany), the fluorescence intensities were quantified using Image J, and the corrected fluorescence intensities were presented, as described previously (46 (link)).

Colabeling of TUNEL and insulin was performed by using a Click-iT Plus TUNEL Assay kit (C10618, Invitrogen, USA) according to the manufacturer’s instructions with a slight modification. Pancreatic sections were deparaffinized and rehydrated. Then slides were fixed in 4% paraformaldehyde for 15 min, followed by permeabilization with proteinase K. Slides were rinsed in PBS for 5 min and immersed slides in 4% paraformaldehyde for 5 min at 37 °C. Then, TdT buffer was added to pancreatic sections and incubated at 37 °C for 10 min. Then, slides were incubated with TdT reaction mixture and rabbit anti-insulin antibody (1:400, 3014s, Cell Signaling Technology) at 4 °C overnight. Then, slides were rinsed with 3% BSA and 0.1% Triton X-100 in PBS. Click-It Plus TUNEL reaction cocktail and Donkey anti-rabbit IgG H&L (Alexa Fluor® 488) (1:200, Ab150073, Abcam) were added to sections and incubated for 30 min at 37 °C protected from light. Then, slides were mounted in DAPI with mounting medium.

Kidney grafts were collected from the recipient mice, and embedded in OCT (optimal cutting temperature) compound (Tissue-Tek, Sakura Finetek, CA). Sections were cut at 7 µm thickness, and blocked with 0.3% hydrogen peroxide.
To measure graft volume, every 6^th section was stained for insulin using rabbit anti-insulin antibody (Cell Signaling, Danvers, MA), anti-rabbit secondary antibody (DAKO, Carpinteria, CA), a peroxidase substrate containing 3,3-diaminobenzidine (DAB-brown) in a DAKO IHC autostainer. Sections were counterstained with hematoxylin on a Leica autostainer XL before mounting with MM 24 (Leica Biosystems, Nussloch, Germany) and a Leica auto-coverslip machine CV5030. Images were taken with a Leica DFC 450 camera on a Leica DM 4000 microscope.