May 02, 2022
Immunofluorescent staining of pancreatic sections
- Caroline CT Tremblay1,
- Valentine Moullé1,
- Bader Zarrouki1,
- Julien Ghislain1,
- Vincent Poitout2
- 1Montreal Diabetes Research Center, CRCHUM, Montréal, QC, Canada.;
- 2Montreal Diabetes Research Center, CRCHUM, and Department of Medicine, Université de Montréal, Montréal, QC, Canada.
Protocol Citation: Caroline CT Tremblay, Valentine Moullé, Bader Zarrouki, Julien Ghislain, Vincent Poitout 2022. Immunofluorescent staining of pancreatic sections. protocols.io https://dx.doi.org/10.17504/protocols.io.bg6ujzew
Manuscript citation:
Glucose and fatty acids synergistically and reversibly promote beta cell proliferation in rats. Moullé VS, Vivot K, Tremblay C, Zarrouki B, Ghislain J, Poitout V. Diabetologia. 2017 May;60(5):879-888. doi: 10.1007/s00125-016-4197-8. Epub 2017 Jan 11. PMID: 28078385. Epidermal growth factor receptor signaling promotes pancreatic β-cell proliferation in response to nutrient excess in rats through mTOR and FOXM1. Zarrouki B, Benterki I, Fontés G, Peyot ML, Seda O, Prentki M, Poitout V. Diabetes. 2014 Mar;63(3):982-93. doi: 10.2337/db13-0425. Epub 2013 Nov 5. PMID: 24194502.
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: June 04, 2020
Last Modified: May 02, 2022
Protocol Integer ID: 37812
Keywords: immunohistochemistry, pancreas
Funders Acknowledgement:
CIHR
Grant ID: 0406014428
NIH
Grant ID: 5R01DK058096-15
Abstract
This protocol describes the steps for performing fluorescence immunohistochemistry on fixed-frozen pancreatic tissue sections. It is suitable for pancreatic tissue isolated from rats and mice at postnatal to adult stages. We routinely apply this protocol to quantify total and proliferating or apoptotic beta-, alpha- and delta-cells. Briefly, pancreata are fixed in 4% paraformaldehyde solution and cryoprotected overnight in 30% sucrose. Tissue are then embedded, frozen, sectioned and mounted on slides. Antigen retrieval is performed using sodium citrate buffer prior to immunostaining.
Image Attribution
Caroline Tremblay
Guidelines
The slides should not be allowed to dry during the staining steps. Drying out will cause nonspecific antibody binding and therefore high background staining.
Materials
MATERIALS
Hoechst 33342, Trihydrochloride, Trihydrate - 10 mg/mL Solution in WaterInvitrogen - Thermo FisherCatalog #H3570
OCT (Optimal Cutting Temperature compound)Sakura FinetekCatalog #4583
VECTASHIELD® Hardset™ Antifade Mounting MediumVector LaboratoriesCatalog #H-1400
Phosphate Buffered Saline 10x (solution)Bio Basic Inc.Catalog #PD8117
Paraformaldehyde 10% bufferedNewcomer SupplyCatalog #13301A
Donkey serumSigma AldrichCatalog #S30-100ML
Triton X-100Sigma AldrichCatalog #T9284
Dako Pen Delimiting penAgilent TechnologiesCatalog #S2002
Bovin serum albuminSigma AldrichCatalog #A7888
Sucrose Ultra PureBioshopCatalog #SUC507.5
Sodium citrate crystals reagentACP ChemicalsCatalog #S2990
Superfrost Plus Microscope SlidesFischer ScientificCatalog #12-550-15
MilliQ waterContributed by users
Equipment
Surgipath® Clear Disposable Base Molds
NAME
Leica
BRAND
75809-376
SKU
Equipment
Well, 250ml. w/ lid, green, xylene resistant
NAME
TBS
BRAND
SS-WLG
SKU
Safety warnings
When working with PFA, always work in the chemical hood.
Before start
Make sure you have the right combination of secondary and primary antibodies with regards to the host species and the fluorophore.
Preparation of cryosections
Preparation of cryosections
1d
1d
Tissue fixation
Safety information
When working with PFA, always work in a chemical hood.
Harvest the pancreas and place it in a 50 ml Falcon tube containing 30 mL of cold 4 % PFA (12 mL of 10 % PFA + 18 mL of PBS).
Fix for 04:00:00 at 4 °C in the dark.
Then, working in a chemical hood, delicately remove the pancreas with forceps and place it on brown paper to absorb the fixative.
Place the organ in a new 50 ml Falcon tube containing 30 mL of a 30 % sucrose solution (9 g of sucrose and 30 mL of PBS).
Store15:00:00 at 4 °C in the dark.
The next day, delicately remove the pancreas with forceps and place it on brown paper to absorb the sucrose solution. Place it in a mold, cover with OCT and freeze at -80 °C until ready for sectioning.
19h
Preparing cryosections
Set the cryostat and the pedestal temperature at -20 °C .
Gather all of the needed material (OCT, slides, pencil, blades, paintbrushes, aluminium foil, slide box, tissues).
Cut cryosections at 0.8 µm thickness and collect on Superfrost Plus microscopic slides.
Store the sections at -80 °C until ready for staining.
2h
Immunofluorescent staining
Immunofluorescent staining
1d
1d
Antigen retrieval
Bring the slides toRoom temperature by putting them in the slide holder in PBS until ready to perform antigen retrieval step.
Put the holder in a 250 ml well filled with milliQ water for 00:05:00 at Room temperature .
Transfer the slides to the sodium citrate solution (10 millimolar (mM) 6 ).
Heat the slides in the microwave with the following sequence:
Heat 00:03:00 (if the solution starts to boil, stop heating and add the remaining time to the pause section; the antigen retrieval step must last a total of 00:20:00 ).
Remove from the microwave and let stand 00:17:00 .
Heat 00:01:00 and let cool down for 00:30:00 at Room temperature .
Rinse the slides in milliQ water for 00:05:00 .
Transfer slides to PBS for 00:05:00 .
1h
Blocking
Dry the slides and outline the tissue with a hydrophobic barrier pen.
Place the slides with the tissue facing up in an humidified chamber and add a sufficient volume of blocking solution (0.1 % (v/v) Triton, 1 % (v/v) BSA plus 5 % (v/v) normal donkey serum in PBS). Use between 150 µL -300 µL depending on the size of the tissue.
Block for 01:00:00 at Room temperature .
1h
Primary antibody staining
Note
At no time should the slides be allowed to dry. Drying out will cause nonspecific antibody binding and therefore high background staining.
Remove the blocking solution and replace with 150 µL -300 µL antibody mix diluted in blocking solution.
Incubate in the humidified chamber 15:00:00 at 4 °C .
15h
Secondary antibody staining
The following morning decant the primary antibody mixture and wash the slides three times in PBS for00:05:00 using a slide holder and well.
Return slides to the humidified chamber and incubate with the secondary antibody diluted in blocking solution for 01:00:00 at Room temperature .
Remove the antibody mixture and wash three times 00:05:00 in PBS.
1h 30m
Hoechst staining and mounting
Label nuclear DNA by incubating the slides in the Hoechst solution (10 µL of Hoechst in 250 mL of PBS) for 00:10:00 at Room temperature using slide holder and well.
Wash the slides three times 00:05:00 in PBS.
Mount slides using VECTASHIELD HardSet Mounting Medium without DAPI.
Place the mounted slides in a cardboard slide tray holder in the dark until ready to acquire the images with a fluorescent microscope.
30m