Islet Preparation

Islet Isolation/Harvest from Rat Pancreas






External Links

JoVE: A Method for Murine Islet Isolation and Subcapsular Kidney Transplantation

[pdf] Written Protocol for Islet Isolation



Islet Fixation and Embedding


Fixation

Reagents needed (all ice cold):
          - 4% Paraformaldehyde (PFA) for regular IHC
          - PBS

Procedure:
1. Pick ~50 Islets in 1.5 mL Eppendorf tube
2. Add 1 mL cold PBS
3. Centrifuge at 1.5g for 5 min, 4oC
4. Add 1 mL cold PFA
5. Incubate at 4oC for 2-24 hours.
6. Remove fixative
7. Add 1 mL cold PBS
8. Keep at 4oC overnight, or can let sit for a few days.

Embedding

Reagents needed:
          - HistoGel (Richard Allan Scientific, Fisher #22-045381)
          - Affi-Gel Blue Gel 150-300 µm (BioRad #153-7301)
          - Tissue-Loc cassette Histoscreen (Richard Allan Scientific, Fisher #22-045346)
          - 19 Ga. long needle
          - PBS

Procedure:
1. Warm up HistoGel at 60oC for about 1 hour
2. Transfer islets into 0.5 mL tube, put on ice.
3. Take Affi-Gel Blue beads from 4oC
          - to prepare beads, pipet some in 1 mL PBS, mix, spin 1 min @2000 rpm or let settle, wash twice, then leave in PBS
4. Cut 10 µL micropipette tip with blade, pipet 10-20 beads into tube with islets
5. Spin islets and beads down at high speed for 10 sec
6. Remove PBS with 10 µL micropipet tip
7. Take HistoGel from water bath to a beaker containing warm water (prevents HistoGel solidification)
8. Add 100 µL HistoGel to islets and beads, pipet up-down once to mix.
9. Immediately spin down at high speed for 1 min.
          - if islets and beads are not in the bottom of the tube, put tube in 60oC water bath for 10 min, then spin again.
10. Put tube on ice for 10 min.

For Paraffin Embedding

11. Mark Tissue-Loc cassettes with pencil
12. Use 19 Ga. long needle to circle HistoGel, drop off gel on a tray.
13. Cut off islet-bead pellet and put on Tissue-Loc Casette, close.
14. Submerge casette in 70% EtOH in a beaker, put at 4oC.
15. Processing (1 hour each solution), embed in paraffin following protocol for whole pancreas from steps 1-6 for Paraffin embedding