TITLE: Assessment of Human Islet Composition and Acinar Cell Component by Immunofluorescence Staining
AUTHORS: IIDP-HIPP 

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;font-style:italic;">This Standard Operating Procedure (SOP) is based on the Vanderbilt Human Islet Phenotyping Program Core Facility (VHIPP) Immunofluorescence Staining Procedure. This SOP provides HIPP procedure for immunofluorescent staining, imaging, and analysis of islet preparations.</span></div><div class = "text-block">This SOP defines the assay method used by the Human Islet Phenotyping Program (HIPP) for quantitative and qualitative determination of the Purified Human Pancreatic Islet product, post-shipment, manufactured for use in the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)-sponsored research in the Integrated Islet Distribution Program (IIDP).</div></div>

[STEPS]
?. [Preparation of Reagents]
Preparation of Reagents for Immunofluorescence Staining of Islet Cryosections
?. [Preparation of Reagents]
10% Triton X-100 stock () – combine  Triton-X-100 and  1X PBS. Mix on shaker for 30 min or until Triton X-100 is completely dissolved and store at for up to 1 month.
30 mL
3 mL
27 mL
4 °C
?. [Preparation of Reagents]
Permeabilization Solution (0.2% Triton,  ) – combine of 10% Triton stock and  1X PBS.
50 mL
1 mL
49 mL
?. [Preparation of Reagents]
Blocking Buffer (5% NDS, ) – combine NDS and 1X PBS.
4 mL
0.2 mL
3.8 mL
?. [Preparation of Reagents]
Antibody Buffer () – combine  BSA,  10% Triton stock, and  1X PBS.
10 mL
0.1 g
0.1 mL
9.8 mL
?. [Preparation of Reagents]
DAPI staining solution (1:25,000,  ) – combine  DAPI stock (5mg/mL) and  1X PBS.
50 mL
2 µl
50 mL
?. [Procedure]
Immunofluorescence Staining Procedure on Islet Cryosections
?. [Procedure]
Use freshly-made antibody incubation buffers, and wash buffers. Steps 2.3, 2.4, 2.8, 2.10, 2.11 can be done in Kartell Staining Chambers.
?. [Procedure]
Let the frozen sections thaw at room temperature and air-dry for about 30 minutes.
?. [Procedure]
Wash the sections with  1X PBS 3 times for 5 minutes to remove the OCT.
50 mL
?. [Procedure]
Permeablize the tissue section with 0.2 % Triton for 15 minutes at room temperature.
?. [Procedure]
Wash the tissue in  1X PBS 3 times for 3-5 minutes.
50 mL
?. [Procedure]
Draw circles or rectangles around the sections with PAP marker and let them dry for about 5 minutes.
?. [Procedure]
Block the sections with 5% normal donkey serum (made from 100% stock)/1X PBS at room temperature for 90 minutes in a humidified chamber.
?. [Procedure]
Aspirate the blocking solution, add primary antibodies (Table 1) diluted in 0.1% Triton-X-100 (made from 10% Triton stock)/1% BSA/1X PBS and incubate in a humidified chamber overnight at  .Table 1. List of primary and secondary antibodies for assessment of islet cell composition and endocrine/acinar cell composition
4 °C
Antibody InformationPrimary Antibodies:C-peptide (rat): GN-ID4 RRID:AB_2631151Glucagon (mouse): ab10988 RRID:AB_297642Glucagon (rabbit): 2760S RRID:AB_659831Somatostatin (goat): sc-7819 RRID:AB_2302603HPX1 (mouse): NBP1-18951, RRID:AB_1625456Secondary Antibodies:Rat IgG-Cy2 (donkey): 712-225-150 RRID:AB_2340673Rat IgG-Cy5 (donkey): 712-175-150 RRID:AB_2340671Mouse IgG-Cy3 (donkey): 715-165-150 RRID:AB_2340813Rabbit IgG-Cy5 (donkey): 711-175-152 RRID:AB_2340607Goat IgG-Cy5 (donkey): 705-175-147 RRID:AB_2340415Mouse IgG-Cy3 (donkey): 715-165-150 RRID:AB_2340813
?. [Procedure]
Aspirate the primary antibodies and wash the sections with 1X PBS three times for 10 minutes each.
?. [Procedure]
Add secondary antibodies (Table 1) diluted in 0.1% Triton/1% BSA/1X PBS and incubate for 1.5 hours at room temperature in a humidified chamber.
?. [Procedure]
Aspirate the secondary antibody and counterstain slides with 1:25,000 DAPI/PBS for 10 minutes at room temperature.
?. [Procedure]
Remove from DAPI and wash the sections with 1X PBS three times for 15 minutes each.
?. [Procedure]
Mount the sections with SlowFade Gold mounting medium.
?. [Imaging and Analysis]
Imaging and Analysis of Fluorescently Labeled Islet Cryosections
?. [Imaging and Analysis]
Capture images of islet sections using a high-resolution whole slide scanning system (ScanScope FL, Aperio/Leica) connected to a web-based digital slide repository powered by eSlide Manager and housed in the Vanderbilt University Medical Center data center (examples of islet images are shown in Figures 1 and 2).
?. [Imaging and Analysis]
Using a tissue classifier algorithm (Halo™, Indica Labs) analyze islet images (50 -100 islets/labeling experiment) to provide a quantitative assessment of the islet cell composition and endocrine/acinar cell compartments for a given human islet preparation (examples of quantitative islet assessment are shown in the legend of Figures 1 and 2).
?. [Data Storage and Reporting]
Data Storage and Reporting
?. [Data Storage and Reporting]
To facilitate data management and ensure data security, the Vanderbilt HIPP uses an institutional server-based platform for data storage and analysis.
?. [Data Storage and Reporting]
Upon analysis completion (within 14 business days) annotated images containing metadata and image analysis outputs will be uploaded to the IIDP-HIPP database and immediately disseminated to IIDP-affiliated investigators and islet isolation centers.