and equipment: Fixation
- surgical instruments for pancreas dissection
- 4% PFA
- rotating "Nutator"
- 70% EtOH
- Weigh mouse
- Euthanize mouse utilizing CO2 and immediately remove
- Weigh the pancreas (dry)
- Fix in 4% PFA for 4 hr at 4°C on a rotating Nutator
- Wash 2 x 15 min in 1X PBS at 4°C on a rotating Nutator
- Incubate O/N at 4°C in 70% EtOH
- Embed in paraffin
- Starting in the middle of the block, section pancreas in 5 μm
section placing 5 sections on each slide. Prepare 100 slides
(or 500 sections).
- Select every 10th slides for
immunohistochemistry. There should be at least 250 μm between
- Select one section per slide for IHC cell mass
and equipment: Immunohistochemistry
- PBS-T (0.2% Tween 20 in PBS)
- 100%, 95%, 70% EtOH, MeOH and 30% H2O2 in
- Dako Cytomation pen
- Vectastain Elite ABC kit (Standard*)
- DAB Peroxidase Substrate kit (Vector)
- Nikon scanner
- Metamorph software
- De-paraffin in a fume hood using slid jars/holders
- Submerge 2 x 5 min in xylene
- Submerge 2 x 5 min in 100% EtOH
- Submerge 1 x 5min in MeOH
- Incubate 1 x 30 min in MeOH/Peroxide (100 ml MeOH + 4ml 30%
- Submerge 2 x 5 min in 95% EtOH
- Submerge 1 x 5 min in 70% EtOH
- Submerge 1 x 5 min in tap H2O
- Submerge 3 x in PBS
- Circumscribe the section using a Pap pen
- Block by incubating sections in 1% BSA + 5% NS (normal serum)
in PBS-T for 1hr X 30 min at RT.
- Incubate sections with anti-insulin antibody at 1:1000 in 1%
BSA + 5% NDS in PBS-T O/N at 4°C.
- Wash sections 2 x 5 min in PBS-T, then 1 x 5 min in PBS.
- Incubate sections with biotin coupled secondary antibody for 30
min at RT.
- Wash 2 x in PBS
- Using the reagents from the Vectastain kit, incubate sections
with the Avidin-HRP (1 ml PBS + 10 µl solution A [Avidin DH] + 10
µl solution B [biotinylated enzyme]) for 30 min at RT (prepare the
A + B complex at least 30 min prior to incubation).
- Wash 2 x with the appropriate buffer.
- Prepare DAB with reagents from Vector DAB kit as follows: 5 ml
DI H2O + 2 drops buffer mix + 4 drops DAB mix + 2 drops
- Incubate with the proper substrate and monitor the staining
under the microscope until color is at the proper intensity.
- Wash 2 x 5 min in H2O
- Counter stain with eosin (dip < 15 sec)
- Incubate 2 x 5 min 70% EtOH
- Incubate 2 x 5 min 95% EtOH
- Incubate 2 x 5 min 100% EtOH
- Dip in xylene and mount with the Permount (Vector).
If secondary antibody is linked to HRP incubate sections with
HRP coupled secondary antibody and omit steps 16 and 17.
- Chose sections in the same position on each slide (for
instance, 2nd section in every slide).
- Scan the entire section on the Nikon scanner
- Erase the "noise" on the image (such as adipose tissue or lymph
- Open the image (minus noise) in Metamorph and measure the
entire area of the pancreas (eosin stained).
- Next, measure the stained islet area (brown) . Export the
data into an Excel spreadsheet to calculate the ratio of stained
islet to whole pancreas.
- Perform these same measurements on 10 sections and calculate
the average ß cell mass by multiplying the islet:pancreas area
ratio X the pancreatic weight.