Dec 06, 2021
Version 2
Assessment of Human Islet Composition and Acinar Cell Component by Immunofluorescence Staining V.2
- IIDP-HIPP1
- 1Integrated Islet Distribution Program and Human Islet Phenotyping Program
- Integrated Islet Distribution Program and Human Islet Phenotyping ProgramTech. support email: amber.m.bradley@vumc.org
Protocol Citation: IIDP-HIPP 2021. Assessment of Human Islet Composition and Acinar Cell Component by Immunofluorescence Staining. protocols.io https://dx.doi.org/10.17504/protocols.io.b2nfqdbnVersion created by Heather Durai
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: December 06, 2021
Last Modified: October 02, 2023
Protocol Integer ID: 55719
Keywords: Human Islet Composition, Acinar Cell Component, Immunofluorescence Staining, HIPP, IIDP,
Abstract
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).
Note
This Standard Operating Procedure (SOP) #: HIPP-07-v02
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.
References:
CITATION
CITATION
Materials
- PBS (phosphate buffered saline) with no Ca/Mg, 1X (Invitrogen 14190-144)
- BSA (bovine serum albumin, Sigma A-6003)
- SlowFade Gold (Molecular Probes S36938)
- Triton X-100 (BioRad 1610407)
- Normal Donkey Serum (NDS, Jackson Immuno Research 017-000-121)
- 4',6-diamidino-2-phenylindole (DAPI, ThermoFisher Scientific D1306)
- Kartell Staining Chambers (VWR 25460-907)
- PAP Marker (Research Products International 195506)
- 4 °C Refrigerator (ArcticTemp)
PBS (phosphate buffered saline) with no Ca/Mg 1X Thermo Fisher ScientificCatalog #Invitrogen 14190-144
BSA (bovine serum albumin)Sigma AldrichCatalog #A-6003
SlowFade Gold (Molecular Probes)Thermo Fisher ScientificCatalog #S36938
Triton X-100Bio-rad LaboratoriesCatalog #1610407
Normal Donkey SerumJackson ImmunoresearchCatalog #017-000-121
4, 6-diamidino-2-phenylindole (DAPI)Thermo Fisher ScientificCatalog #D1306
Equipment
Kartell Staining Chambers
NAME
VWR
BRAND
25460-907
SKU
LINK
Equipment
Marker
NAME
PAP
BRAND
195506
SKU
LINK
Equipment
4°C Refrigerator
NAME
ArcticTemp
BRAND
None
SKU
LINK
Safety warnings
Triton X-100 (BioRad 1610407)
Safety information
Triton X-100.pdf Preparation of Reagents
Preparation of Reagents
Preparation of Reagents for Immunofluorescence Staining of Islet Cryosections
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 °C for up to 1 month.
Permeabilization Solution (0.2% Triton, 50 mL ) – combine 1 mL of 10% Triton stock and 49 mL 1X PBS.
Blocking Buffer (5% NDS, 4 mL ) – combine 0.2 mL NDS and 3.8 mL 1X PBS.
Antibody Buffer (10 mL ) – combine 0.1 g BSA, 0.1 mL 10% Triton stock, and 9.8 mL 1X PBS.
DAPI staining solution (1:25,000, 50 mL ) – combine 2 µL DAPI stock (5mg/mL) and 50 mL 1X PBS.
Procedure
Procedure
Immunofluorescence Staining Procedure on Islet Cryosections
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.
Let the frozen sections thaw at room temperature and air-dry for about 30 minutes.
Wash the sections with 50 mL 1X PBS 3 times for 5 minutes to remove the OCT.
Permeablize the tissue section with 0.2 % Triton for 15 minutes at room temperature.
Wash the tissue in 50 mL 1X PBS 3 times for 3-5 minutes.
Draw circles or rectangles around the sections with PAP marker and let them dry for about 5 minutes.
Block the sections with 5% normal donkey serum (made from 100% stock)/1X PBS at room temperature for 90 minutes in a humidified chamber.
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
Note
Antibody Information
Primary Antibodies:
Secondary Antibodies:
Aspirate the primary antibodies and wash the sections with 1X PBS three times for 10 minutes each.
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.
Aspirate the secondary antibody and counterstain slides with 1:25,000 DAPI/PBS for 10 minutes at room temperature.
Remove from DAPI and wash the sections with 1X PBS three times for 15 minutes each.
Mount the sections with SlowFade Gold mounting medium.
Imaging and Analysis
Imaging and Analysis
Imaging and Analysis of Fluorescently Labeled Islet Cryosections
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).
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).
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.
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.
Citations
Dai C, Brissova M, Hang Y, Thompson C, Poffenberger G, Shostak A, et al. Islet-enriched gene expression and glucose-induced insulin secretion in human and mouse islets
https://pubmed.ncbi.nlm.nih.gov/22167125/Guo S, Dai C, Guo M, Taylor B, Harmon JS, Sander M, et al. Inactivation of specific β cell transcription factors in type 2 diabetes
https://pubmed.ncbi.nlm.nih.gov/23863625/