TITLE: Human Pancreas Optical Clearing by iDISCO and VISIKOL 
AUTHORS: Martha Campbell Thompson, Malavika Nair

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Optical clearing of tissues to improve deep tissue microscopy was described over a century ago and methods have improved considerably following publication of the CLARITY method from Karl Deisseroth in 2013. Improvements in organic and inorganic methods are constantly reported for multiple species and organs. Large volume imaging was needed for human pancreas studies due to known heterogeneity in normal islet sizes and proportions of endocrine cells. Lobularity in islet beta-cell loss and islet inflammation is also seen in patients with type 1 diabetes. The </span><a href="http://www.idisco.info" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">iDISCO</span></a><span> protocol allows for rapid clearing, easy staining, and high resolution confocal imaging. Human pancreas samples were cleared using </span><a href="http://www.idisco.info" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">iDISCO</span></a><span> as originally published. This protocol describes steps to clear 4mm x 6mm x 400um deep sections of 4% paraformaldehyde fixed human pancreas using iDISCO-like reagents purchased from </span><a href="https://visikol.com/" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Visikol</span></a><span>. </span></div></div>

[STEPS]
?. [Permeabilization]
On a shaker, wash the fixed tissue 2x30 minutes in 1X PBS at room temperature.
?. [Permeabilization]
Dehydrate the tissue in increasing grades of methanol, 50%-80%-100% MeOH, for 1 hour each at room temperature.
?. [Permeabilization]
Place the tissue in 5% H2O2/20% DMSO/MeOH overnight on a shaker at room temperature.
?. [Permeabilization]
Wash tissue 2x1 hour in 20% DMSO/MeOH at room temperature.
?. [Permeabilization]
Wash tissue in decreasing grades of methanol, 100%- 80%-50% MeOH, for 1 hour each at room temperature.
?. [Permeabilization]
Place tissue in 1X PBS with 1% TritonX-100 for 30 minutes at room temperature.
?. [Labeling]
Place the samples in Penetration Buffer for 36 hours at room temperature.
?. [Labeling]
Transfer the samples to Blocking Buffer and incubate for 36 hours at room temperature.
?. [Labeling]
Using the Antibody solution as a diluent, prepare the primary antibody cocktail and incubate the samples for 48 hours at room temperature.Primary and secondary antibodies are listed in the JOVE publication.We utilized cocktails of primary and secondary antibodies, combining primaries raised in mouse, rabbit, goat, and chicken and suitable secondary antibodies raised in donkey (ThermoFisher, Jackson Immunoresearch).
?. [Labeling]
Wash the samples in the Wash Buffer 5x30 minutes at room temperature.
?. [Labeling]
Using the Antibody solution as a diluent, prepare the secondary antibody cocktail and incubate the samples for 48 hours at room temperature.
?. [Labeling]
Wash the samples in the Wash Buffer 5x30 minutes at room temperature.
?. [Clearing]
Wash the tissue in increasing grades of methanol,  50%-80%-100% MeOH, for 1 hour each at room temperature.
?. [Clearing]
Incubate the samples in HISTO-1 solution for 16 hours at room temperature.
?. [Clearing]
Incubate the samples in HISTO-2 solution for 16 hours at room temperature.
?. [Imaging]
Mount samples on glass slides in HISTO-2 with a #1.5 coverslip using silicon spacers or use glass chamber slides or small imaging dish.
?. [Imaging]
Image using a confocal microscope or suitable Lightsheet.