Islet Preparation
Islet Isolation/Harvest from Rat PancreasExternal 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:
- Pick ~50 Islets in 1.5 mL Eppendorf tube
- Add 1 mL cold PBS
- Centrifuge at 1.5g for 5 min, 4oC
- Add 1 mL cold PFA
- Incubate at 4oC for 2-24 hours.
- Remove fixative
- Add 1 mL cold PBS
- 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:
- Warm up HistoGel at 60oC for about 1 hour
- Transfer islets into 0.5 mL tube, put on ice.
- 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
- Cut 10 µL micropipette tip with blade, pipet 10-20 beads into tube with islets
- Spin islets and beads down at high speed for 10 sec
- Remove PBS with 10 µL micropipet tip
- Take HistoGel from water bath to a beaker containing warm water (prevents HistoGel solidification)
- Add 100 µL HistoGel to islets and beads, pipet up-down once to mix.
- 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.
- Put tube on ice for 10 min.
For Paraffin Embedding
- Mark Tissue-Loc cassettes with pencil
- Use 19 Ga. long needle to circle HistoGel, drop off gel on a tray.
- Cut off islet-bead pellet and put on Tissue-Loc Casette, close.
- Submerge casette in 70% EtOH in a beaker, put at 4oC.
- Processing (1 hour each solution), embed in paraffin following protocol for whole pancreas from steps 1-6 for Paraffin embedding