Morphological staining
After the OGT2115 administration, the mice were euthanized with
CO2. The pancreas was removed and fixed in 4%
neutral-buffered formalin and processed for paraffin embedding (Sunet al. , 2018). Sections were stained with Alcian blue (Sigma,
0.2% in 0.2 mol/L phosphate buffer, 0.65 mol/L MgCl2,
pH 5.8) and counterstained with safranin O (Solarbio, Beijing, China).
For immunostaining, the sections were immersed in citrate buffer (0.01
M, pH=6.0) at 95 °C for 5 min to perform heat-induced antigen retrieval.
The primary antibodies used were listed in Table S1. Especially, before
using 3G10 antibody [US Biological Cat#H1890-75, RRID:
AB_2722745], 1 mU heparitinase I (Amsbio, Madrid, Spain) was
incubated to exposure 3G10 epitope of HS. For immunohistochemistry, DAB
kits (Gene Tech, Shanghai, China) were used to show the
immunohistochemical staining and the nuclei were stained with
hematoxylin. Images were obtained with a DP70 camera (Olympus, Tokyo,
Japan). Image J software [RRID: SCR_003070] (V1.8.0, NIH, Bethesda,
MD, USA) was used to calculate the intensity of the immunohistochemical
staining, expressed based on islet area.
For immunofluorescence, the secondary antibodies were: donkey
anti-mouse/rabbit/Guinea Pig IgG conjugated with AlexaFluor 488/594
(Invitrogen, Carlsbad, CA, USA). The nuclei were stained with DAPI
(Sigma). Apoptotic cells were stained using a TdT-mediated dUTP Nick-End
Labeling (TUNEL) kit (Vazyme Biotech, Nanjing, China) following the
manufacturer’s protocol. The fluorescence images were scanned using a
confocal microscope (Olympus F1000).