Aug 14, 2020
Human Islet Microvasculature Immunofluorescence in Optically Cleared Samples
- Martha Campbell-Thompson1,
- Elizabeth Butterworth Hosaka2,
- Katelyn N Carty2
- 1University of Florida;
- 2co-author
- Human BioMolecular Atlas Program (HuBMAP) Method Development CommunityTech. support email: Jeff.spraggins@vanderbilt.edu
External link: https://www.protocols.io/view/human-pancreas-pact-optical-clearing-and-high-reso-9gbh3sn/materials
Protocol Citation: Martha Campbell-Thompson, Elizabeth Butterworth Hosaka, Katelyn N Carty 2020. Human Islet Microvasculature Immunofluorescence in Optically Cleared Samples . protocols.io https://dx.doi.org/10.17504/protocols.io.y3tfynn
Manuscript citation:
Butterworth E, Dickerson WD, Vijay V, Weitzel K, Cooper J, Atkinson EW, Coleman JE, Otto KJ, Campbell-Thompson M. High resolution 3D Imaging of the Human Pancreas Neuro-Insular Network. J Vis Exp. 2018 Jan 29;(131). doi: 10.3791/56859.2018. PMID: 29443037.
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: March 12, 2019
Last Modified: August 14, 2020
Protocol Integer ID: 21331
Keywords: human islet, glucagon, optical clearing, confocal microscopy, 3D, PACT, collagen IV, SMA, Vesselucida360, alpha-cells, pancreas, neuro-insular network, passive CLARITY
Disclaimer
Different primary and secondary antibody lots may differ in affinities and should be independently optimized.
Abstract
This protocol describes the immunostaining performed on five human control pancreas samples, cleared using a modified passive CLARITY (PACT) method according to our published methods. Islets were identified based on alpha-cells stained with glucagon while collagen-containing basement membranes in the extracellular matrix were stained using anti-collagen IV. Smooth muscle cells surrounding arteries and arterioles were identified using anti-SMA primary antibody directly conjugated with Cy3 fluorophore. An accompanying protocol describes using Vesselucida360 to contour islets and determine islet basement membrane and SMA densities and morphometric variables (https://dx.doi.org/10.17504/protocols.io.bjfzkjp6).
Attachments
Image Attribution
An islet is shown from case 6232 with all 3 fluorescent channels visible.
Guidelines
This protocol is for PACT-cleared human pancreas samples, fixed with 4% paraformaldehyde for at minimum 48:00:00 , 4% agarose embedded and sectioned at 400 um or greater thickness. The Triton-X concentration in the antibody diluent was increased from 0.1% to 0.5% based on experiments that showed antibody penetration into the middle of the sample was improved without loss of signal.
Materials
MATERIALS
Glucagon (GCG) Mouse anti-humanAbcamCatalog #ab10988 (Lot# GR290488-2)
Goat anti-ms Dylight 405 hi-crossThermo Fisher ScientificCatalog #35500BID
PBS Phosphate Buffered Saline 10X SolutionFisher ScientificCatalog #BP399-1
Goat normal serumVector LaboratoriesCatalog #S-1000
Triton X-100Merck MilliporeSigma (Sigma-Aldrich)Catalog #T8787-100
Collagen IV (Col14) Rabbit anti-humanAbcamCatalog #ab6586
Smooth muscle actin- alpha (a-SMA) Mouse anti-human-Cy3 [clone 1A4] Merck MilliporeSigma (Sigma-Aldrich)Catalog #C6198
Goat anti-rb AF488 hi crossThermo Fisher ScientificCatalog #A-11034
STEP MATERIALS
Triton X-100Merck MilliporeSigma (Sigma-Aldrich)Catalog #T8787-100
PBS Phosphate Buffered Saline 10X SolutionFisher ScientificCatalog #BP399-1
Software
Vesselucida 360
NAME
MBF Bioscience
DEVELOPER
Software
Vesselucida Explorer
NAME
MBF Bioscience
DEVELOPER
Protocol materials
Goat anti-ms Dylight 405 hi-crossThermo Fisher ScientificCatalog #35500BID
PBS Phosphate Buffered Saline 10X SolutionFisher ScientificCatalog #BP399-1
PBS Phosphate Buffered Saline 10X SolutionFisher ScientificCatalog #BP399-1
Triton X-100Merck MilliporeSigma (Sigma-Aldrich)Catalog #T8787-100
Smooth muscle actin- alpha (a-SMA) Mouse anti-human-Cy3 [clone 1A4] Merck MilliporeSigma (Sigma-Aldrich)Catalog #C6198
Glucagon (GCG) Mouse anti-humanAbcamCatalog #ab10988 (Lot# GR290488-2)
Triton X-100Merck MilliporeSigma (Sigma-Aldrich)Catalog #T8787-100
Goat normal serumVector LaboratoriesCatalog #S-1000
Collagen IV (Col14) Rabbit anti-humanAbcamCatalog #ab6586
Goat anti-rb AF488 hi crossThermo Fisher ScientificCatalog #A-11034
PBS Phosphate Buffered Saline 10X SolutionFisher ScientificCatalog #BP399-1
PBS Phosphate Buffered Saline 10X SolutionFisher ScientificCatalog #BP399-1
Goat normal serumVector LaboratoriesCatalog #S-1000
Triton X-100Merck MilliporeSigma (Sigma-Aldrich)Catalog #T8787-100
Safety warnings
Follow all laboratory safety procedures when handling hazardous chemicals and materials.
Before start
Ensure samples are cleared and the SDS is well-removed by extensive washing in PBS at Room temperature on a rocker plate.
For these samples, optical clearing was performed using 4% SDS at 60 °C with rocking for up to 3 weeks until the samples reached transparency. Our original protocol used 8% SDS. We have subsequently tested both 4% and 8% SDS at Room temperature and 60 °C . Different tissues should be tested independently for optimal results.
Prepare the Antibodies
Prepare the Antibodies
Make antibody dilution buffer (ABD): 1x PBS containing 2% normal goat serum (NGS) and 0.5% Triton-X 100.
PBS Phosphate Buffered Saline 10X SolutionFisher ScientificCatalog #BP399-1
Goat normal serumVector LaboratoriesCatalog #S-1000
Triton X-100Merck MilliporeSigma (Sigma-Aldrich)Catalog #T8787-100
Dilute the primary antibodies to 1:200 in ABD and prepare at least 1 mL per sample.
Stain the Tissue
Stain the Tissue
Incubate the samples with primary antibodies on a rocker plate for 48:00:00 1-5 days at Room temperature .
Wash the tissue sections with 1xPBS for five times, minimum01:00:00 at Room temperature .
PBS Phosphate Buffered Saline 10X SolutionFisher ScientificCatalog #BP399-1
Dilute the secondary antibodies to 1:500 and prepare at least 1 mL per sample. Incubate for 48:00:00 or more on a rocker at Room temperature .
Wash the tissue with 1 x PBS 5 times 01:00:00 at Room temperature .
Equilibrate the samples in RIMS containing 0.5% sodium azide for at least 12:00:00 before imaging.
Transfer to an imaging dish or chamber slide using RIMS as mounting media.
Imaging
Imaging
Perform confocal microscopy. Each microscope will require optimization of imaging parameters.
Equipment
LSM 710
NAME
confocal laser microscope
TYPE
Zeiss
BRAND
LSM 710
SKU
LINK
The following lasers were used for imaging each antigen in these images:
Track 1- 488- Collagen IV
Track 2- 405- GCG, 561- SMA
Results
Results
Expected result
Islet alpha-cells- GCG Stain - Specific Stain, Intensity Good, Background Low
Collagen IV- basement membranes- Specific Stain, Intensity Good, Background medium
SMA- smooth muscle cells surrounding vessels - Specific Stain, Intensity Good, Background Low
Analyze islet microvascular density by contouring the GCG+ area for islet volume followed by collagen IV and SMA morphometry using Vesselucida360.