TITLE: Islet Embedding for Histology
AUTHORS: IIDP-HIPP 

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;font-style:italic;">This Standard Operating Procedure (SOP) is based on the Vanderbilt Human Islet Phenotyping Program (HIPP) Immobilization of Islets in 3-D Gel for Histology. This SOP provides HIPP procedure for embedding islets in Collagen I gel for cryosectioning and histological staining. </span></div><div class = "text-block">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).</div></div>

[STEPS]
?. [Procedures]
Islet Immobilization and Fixation
?. [Procedures]
Prepare Collagen I working solution ( ) by combining  Collagen I stock,  sterile water,  HEPES,  NaHCO3,  5X DMEM.
1 mL
375 µl
355 µl
20 µl
50 µl
200 µl
?. [Procedures]
Using a P-1000 pipette and wide-bore tips, transfer an aliquot of islet suspension containing approximately 500 IEQs into a  centrifuge tube and centrifuge at 1000 rpm for 1 min. Aspirate the supernatant. If islet medium is rich in serum, wash the islets once with 1X PBS.
1.5 mL
?. [Procedures]
Add  of Collagen I working solution to the islet pellet and transfer the mixture into a 96-well plate. Place the plate in a tissue culture incubator set to  . Incubate for 90 min and Collagen I will form into a gel. While gel is solidifying, islets will settle by gravity at the bottom of the gel and form a monolayer.
150 µl
37 °C
?. [Procedures]
Place 96-well plate on ice and add ice-cold 4% paraformaldehyde/1X PBS on top of the gel to fill up the well. Fix for 10 min on ice.
?. [Procedures]
Using a 28 G needle ( insulin syringe) loosen up the gel containing islets from the sides of the well. Then, using a fine spatula transfer the gel into a 12-well plate containing  of ice-cold 4% paraformaldehyde/1X PBS per well. Be very careful while you do this because the gel is relatively soft. Fix the islets on ice for an additional 15 min under very mild agitation using an adjustable tilt rocker (low setting).
0.3 mL
3 mL
?. [Procedures]
Gently aspirate the fixative and wash the gel with  of 1X PBS on ice for 20 min under very mild agitation using an adjustable tilt rocker (low setting).
3 mL
?. [Procedures]
Repeat step 1.6 two times.
?. [Procedures]
Islet Embedding and Sectioning
?. [Procedures]
After the last wash, add  of 30% sucrose/1X PBS. Allow the gel to equilibrate for 2-3 hours or overnight at  .
3 mL
4 °C
?. [Procedures]
Lift the gel from the dish using a fine spatula and place it into a standard cryomold that is half-way filled with OCT compound. While doing this, orient the gel such that the bottom of the gel (where the islets are) is facing the bottom of the cryomold. Add more OTC to fill up the cryomold. Take an image of the gel on an Olympus SZX12 microscope at 16x. Example of islets embedded in Collagen I is shown in Figure 1.
?. [Procedures]
Place the embedded gel in a  freezer. When OCT compound is frozen, wrap the block in pre-labeled aluminum foil, place it a zip-lock bag, seal and store at  .
-80 °C
-80 °C
?. [Procedures]
Cut 8-µm cryosections and plate two sections per slide on Plus Gold slides. Transfer slides and blocks on dry ice back to the HIPP and store in a  freezer.
-80 °C
?. [Procedures]
Enter slides and blocks into the Vanderbilt HIPP electronic inventory system and upload images of islet gels to the IIDP-HIPP database.
?. [Deviations and Resolution]
Document any deviations that occurred during this protocol that affect the final results and report with the analysis of the assay.