TITLE: Immunofluorescent staining of pancreatic sections
AUTHORS: Caroline  CT Tremblay, Valentine Moullé, Bader Zarrouki, Julien  Ghislain, Vincent  Poitout

[DESCRIPTION]
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.

[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.

[STEPS]
SECTION: Preparation of cryosections
2. 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.
SECTION: Preparation of cryosections
1. Tissue fixation
 
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 240 min  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). 
Store900 min 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.
SECTION: Immunofluorescent staining
3. Antigen retrieval
Bring the slides to-800 Room 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 5 min at -800 Room temperature.
Transfer the slides to the sodium citrate solution (10 millimolar (mM) pH 6).
Heat the slides in the microwave with the following sequence: 
Heat 3 min (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 20 min).
Remove from the microwave and let stand 17 min. 
Heat 1 min and let cool down for 30 min at -800 Room temperature.
Rinse the slides in milliQ water for 5 min.
Transfer slides to PBS for 5 min.
4. 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 60 min at -800 Room temperature.
5. Primary antibody staining
 
Remove the blocking solution and replace with 150 µL-300 µL antibody mix diluted in blocking solution.
Incubate in the humidified chamber 900 min  at 4 °C.
6. Secondary antibody staining 
The following morning decant the primary antibody mixture and wash the slides three times in PBS for5 min using a slide holder and well.
Return slides to the humidified chamber and incubate with the secondary antibody diluted in blocking solution for 60 min at 40 Room temperature.
Remove the antibody mixture and wash three times 5 min in PBS.
7. 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 10 min at 40 Room temperature using slide holder and well.
Wash the slides three times 5 min 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.