Immunofluorescent staining of pancreatic sections
Caroline CT Tremblay, Julien Ghislain, Vincent Poitout, Valentine Moullé, Bader Zarrouki
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.
Before start
Make sure you have the right combination of secondary and primary antibodies with regards to the host species and the fluorophore.
Steps
Preparation of cryosections
Tissue fixation
30mL of cold 4 % PFA (12mL of 10 % PFA + 18mL of PBS). Fix for 4h 0m 0s 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 30mL of a 30 % sucrose solution (9g of sucrose and 30mL of PBS). Store15h 0m 0s 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. 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.
Immunofluorescent staining
Antigen retrieval Bring the slides to-800Room 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 0h 5m 0s at -800Room temperature.Transfer the slides to the sodium citrate solution (10millimolar (mM) 6).Heat the slides in the microwave with the following sequence: Heat 0h 3m 0s (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 0h 20m 0s).Remove from the microwave and let stand 0h 17m 0s. Heat 0h 1m 0s and let cool down for 0h 30m 0s at -800Room temperature.Rinse the slides in milliQ water for 0h 5m 0s.Transfer slides to PBS for 0h 5m 0s.
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 150µL-300µL depending on the size of the tissue.
Block for 1h 0m 0s at -800Room temperature.
Primary antibody staining
150µL-300µL antibody mix diluted in blocking solution.
Incubate in the humidified chamber 15h 0m 0s at 4°C.
Secondary antibody staining
The following morning decant the primary antibody mixture and wash the slides three times in PBS for0h 5m 0s using a slide holder and well.
Return slides to the humidified chamber and incubate with the secondary antibody diluted in blocking solution for 1h 0m 0s at 40Room temperature.
Remove the antibody mixture and wash three times 0h 5m 0s in PBS.
Hoechst staining and mounting
Label nuclear DNA by incubating the slides in the Hoechst solution (10µL of Hoechst in 250mL of PBS) for 0h 10m 0s at 40Room temperature using slide holder and well.
Wash the slides three times 0h 5m 0s 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.
