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

[DESCRIPTION]
This Standard Operating Procedure (SOP) is based on the Human Islet Phenotyping Program of the IIDP Immunofluorescence Staining Procedure. This SOP provides the HIPP procedure for immunofluorescent staining, imaging, and analysis of islet preparations.

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).


 



[GUIDELINES]
Integrated Islet Distribution Program (IIDP) (RRID:SCR_014387): The IIDP is a grant funded program commissioned and funded by the NIDDK to provide quality human islets to the diabetes research community to advance scientific discoveries and translational medicine. The IIDP consists of the NIDDK Project Scientist and Program Official, the External Evaluation Committee and the CC at City of Hope (COH). The IIDP CC integrates an interactive group of academic laboratories including the subcontracted IIDP centers.

IIDP Coordinating Center (CC): Joyce Niland, Ph.D. and Carmella Evans-Molina, M.D., Ph.D. serve as Co-Principal Investigators (Co-PIs) for the IIDP Program located within the Department of Diabetes and Cancer Discovery Science at COH to coordinate the activities of the IIDP and Human Islet Phenotyping Program (HIPP). Dr. Niland, contact PI, oversees the daily activity of the IIDP staff, provides informatics/ biostatistical input, and subcontracts with the Islet Isolation Centers (IICs) to ensure the delivery of the highest quality human islets to IIDP-approved investigators. Dr. Evans-Molina serves as the liaison to the HIPP, interacting closely to ensure that extensive, high quality phenotypic data are collected on islets distributed by the IICs. She also facilitates the delivery of this information to both the IICs and the IIDP-approved investigators, while responding to questions, issues, or suggestions for further HIPP enhancements.

Human Islet Phenotyping Program (HIPP): The HIPP is a subcontracted entity of the IIDP through the COH and Vanderbilt University. The HIPP is directed by Marcela Brissova, Ph.D. and is responsible for performing specific standardized phenotyping assays agreed upon by both the IIDP and the HIPP, in order to provide enhanced, quality data on the human islets post-shipment, to the IIDP. The results of these assays will be approved by the CC and posted on the IIDP website for both the centers and the approved investigators.

Cryosections: Sections of a tissue/cells embedded in optimal cutting temperature (OCT) compound and frozen -80 °C .

Indirect Immunofluorescence Staining: Immunohistochemical procedure based on antigen detection by flourescence in histological sections using a combination of primary and secondary antibodies where the primary antibody is directed to the antigen of interest and the fluorescently-conjugated secondary recognizes species where the primary antibody was raised. Histological sections are viewed using a microscope system equipped with an appropriate light source and filter set to allow for visualization of fluorescence tissue staining.



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[STEPS]
SECTION: Preparation of Reagents
1. Preparation of Reagents for Immunofluorescence Staining of Islet Cryosections


SECTION: Preparation of Reagents
1.1. 10% Triton X-100 stock (30 mL) – combine 3 mL Triton-X-100 and 27 mL 1X PBS. Mix on shaker for 30 min or until Triton X-100 is completely dissolved and store at 4 °Cfor up to 1 month.
SECTION: Preparation of Reagents
1.2. Permeabilization Solution (0.2% Triton, 50 mL ) – combine 1 mLof 10% Triton stock and 49 mL 1X PBS.
SECTION: Preparation of Reagents
1.3. Blocking Buffer (5% NDS, 4 mL) – combine 0.2 mLNDS and 3.8 mL1X PBS.
SECTION: Preparation of Reagents
1.4. Antibody Buffer (10 mL) – combine 0.1 g BSA, 0.1 mL 10% Triton stock, and 9.8 mL 1X PBS.
SECTION: Preparation of Reagents
1.5. DAPI staining solution (1:25,000, 50 mL ) – combine 2 µL DAPI stock (5mg/mL) and 50 mL 1X PBS.
SECTION: Procedure
2. Immunofluorescence Staining Procedure on Islet Cryosections
SECTION: Procedure
2.1. 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.
SECTION: Procedure
2.2. Let the frozen sections thaw at room temperature and air-dry for about 30 minutes.
SECTION: Procedure
2.3. Wash the sections with 50 mL 1X PBS 3 times for 5 minutes to remove the OCT.

SECTION: Procedure
2.4. Permeablize the tissue section with 0.2 % Triton for 15 minutes at room temperature.
SECTION: Procedure
2.5. Wash the tissue in 50 mL 1X PBS 3 times for 3-5 minutes.

SECTION: Procedure
2.6. Draw circles or rectangles around the sections with PAP marker and let them dry for about 5 minutes.
SECTION: Procedure
2.7. Block the sections with 5% normal donkey serum (made from 100% stock)/1X PBS at room temperature for 90 minutes in a humidified chamber.
SECTION: Procedure
2.8. 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 4 °C .

Table 1. List of primary and secondary antibodies for assessment of islet cell composition and endocrine/acinar cell composition

  

 

 




SECTION: Procedure
2.9. Aspirate the primary antibodies and wash the sections with 1X PBS three times for 10 minutes each.
SECTION: Procedure
2.10. 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.


SECTION: Procedure
2.11. Aspirate the secondary antibody and counterstain slides with 1:25,000 DAPI/PBS for 10 minutes at room temperature.
SECTION: Procedure
2.12. Remove from DAPI and wash the sections with 1X PBS three times for 15 minutes each.
SECTION: Procedure
2.13. Mount the sections with SlowFade Gold mounting medium.
SECTION: Imaging and Analysis
3. Imaging and Analysis of Fluorescently Labeled Islet Cryosections
SECTION: Imaging and Analysis
3.1. 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).

   
  

SECTION: Imaging and Analysis
3.2. 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  Figures 3 and 4).

  

SECTION: Data Storage and Reporting
4. Data Storage and Reporting
SECTION: Data Storage and Reporting
4.1. To facilitate data management and ensure data security, the Vanderbilt HIPP uses an institutional server-based platform for data storage and analysis.
SECTION: Data Storage and Reporting
4.2. 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.