TITLE: Human Islet Microvasculature Immunofluorescence in Optically Cleared Samples 
AUTHORS: Martha Campbell Thompson, Elizabeth Butterworth Hosaka, Katelyn N Carty

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><div class = "justify" style = "text-align:left"><span>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 (</span><a href="https://dx.doi.org/10.17504/protocols.io.bjfzkjp6" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://dx.doi.org/10.17504/protocols.io.bjfzkjp6</span></a><span>). </span></div></div><div class = "text-block"><div class = "justify" style = "text-align:left"></div></div><div class = "text-block"><div class = "justify" style = "text-align:left"></div></div></div>

[STEPS]
?. [Stain the Tissue]
Incubate the samples with primary antibodies on a rocker plate for  1-5 days at  .
0 Room temperature
?. [Stain the Tissue]
Wash the tissue sections with 1xPBS for five times, minimum at .
25 Room temperature
?. [Imaging]
The following lasers were used for imaging each antigen in these images:Track 1-     488- Collagen IVTrack 2-     405- GCG, 561- SMA
?. [Imaging]
Perform confocal microscopy. Each microscope will require optimization of imaging parameters.
?. [Stain the Tissue]
Dilute the secondary antibodies to 1:500 and prepare at least   per sample.  Incubate for  or more on a rocker at .
1 mL
25 Room temperature
?. [Results]
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
?. [Stain the Tissue]
Wash the tissue with 1 x PBS 5 times  at .
25 Room temperature
?. [Results]
Analyze islet microvascular density by contouring the GCG+ area for islet volume followed by collagen IV and SMA morphometry using Vesselucida360.
?. [Prepare the Antibodies]
Make antibody dilution buffer (ABD): 1x PBS containing 2% normal goat serum (NGS) and 0.5% Triton-X 100.
?. For these samples, optical clearing was performed using 4% SDS at  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  and   . Different tissues should be tested independently for optimal results.
60 °C
0 Room temperature
60 °C
?. Dilute the primary antibodies to 1:200 in ABD and prepare at least  per sample.
1 mL
?. [Stain the Tissue]
Equilibrate the samples in RIMS containing 0.5% sodium azide for at least  before imaging. Transfer to an imaging dish or chamber slide using RIMS as mounting media.