Pancreatic tissue or isolated islets were embedded in Tissue-Tek OCT Compound (VWR, Missouri City, TX, USA), and then, snap-frozen. Frozen sections (10 μm) were cut, placed on Superfrost glass slides, and then, stored at −80°C. For immunostaining, sections were fixed in methanol, followed by overnight incubation with primary antibodies raised against insulin (Santa Cruz Biotechnology, Dallas, TX, USA; 1:200 dilution), collagen IV, VI, fibronectin (Abcam, Cambridge, MA, USA; 1:200 dilution), and CD31 (R&D Systems, Minneapolis, MN, USA; 1:100 dilution). Alexa Fluor 647 conjugated secondary antibodies (Abcam; 1:500 dilution) were used for detection.
For immunofluorescence of paraffin embedded rat islets, blocks were cut into 4 μm sections, deparaffinized, rehydrated, and subjected to antigen retrieval with citrate buffer using a microwave oven on high power. Sections were immunostained with anti-insulin (see above) primary antibody followed by Alexa Fluor 488 conjugated antibody (Invitrogen, Carlsbad, CA, USA; 1:1000 dilution).
Both frozen and paraffin sections were mounted in Fluoroshield mounting media containing DAPI (Sigma-Aldrich, St. Louis, MO, USA). Images were captured using an Olympus IX73 fluorescence microscope or Zeiss LSM 710 confocal microscope.
For immunofluorescence of paraffin embedded rat islets, blocks were cut into 4 μm sections, deparaffinized, rehydrated, and subjected to antigen retrieval with citrate buffer using a microwave oven on high power. Sections were immunostained with anti-insulin (see above) primary antibody followed by Alexa Fluor 488 conjugated antibody (Invitrogen, Carlsbad, CA, USA; 1:1000 dilution).
Both frozen and paraffin sections were mounted in Fluoroshield mounting media containing DAPI (Sigma-Aldrich, St. Louis, MO, USA). Images were captured using an Olympus IX73 fluorescence microscope or Zeiss LSM 710 confocal microscope.