TITLE: RNA in situ hybridization on pancreatic sections using RNAscope® technology
AUTHORS: Caroline  CT Tremblay, Julien  Ghislain, Vincent  Poitout

[DESCRIPTION]
This protocol describes the steps for performing RNA in situ hybridization simultaneously for two targets using RNAscope Multiplex Fluorescent reagent Kit v2 assay 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 assess gene expression qualitatively and semi-quantitatively at the cellular level. Briefly, pancreata are fixed in 4% paraformaldehyde solution and cryoprotected overnight in 30% sucrose. Tissue are then embedded, frozen, sectioned and mounted on slides. Tissue section pretreatments, RNAscope probe hybridization and fluorescence detection are performed essentially as described by the manufacturer with minor modifications. 

[BEFORE_START]
Before performing the RNAscope assay, calculate the numbers of slides, you will need (samples + controls), verify that you have enough of each solutions (H2O2, Target retrieval, protease solution, probes, wash buffer, SSC buffer, Amp1-2-3, HRP#C1-C2-C3-C4, Opal dies and mounting media). 

[STEPS]
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: 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   thickness and collect on Superfrost Plus microscopic slides.
Store the sections at -80 °C until ready for staining.
SECTION: Pretreatments
3. Preparation
Turn on the HybEz oven and set to 40 °C.
Place a wet Whatman paper on the Humidity Control Tray.
Insert the covered tray in the HybEz oven and humidify the chamber for at least 30 min.
Wash the slides in a slide holder with PBS for at least 5 min shaking from time to time.
Prepare 1x Target Retrieval solution (1 bottle + 630 mL milliQ water) in a 1 L glass beaker. 
Cover and heat the solution at maximum on a hot plate with constant stirring. Monitor the temperature with an electrical thermometer. When the temperature reaches 98 °C reduce the heat setting to maintain a gentle boil.
Set to boil 700 mLof milliQ water in a 1 L glass beaker that will be used to prewarm the slides (step 5).
SECTION: RNAscope Assay
7. Probe hybridization (2 probe method)
Determine the quantity of probes, transfer to 1.5 ml tubes and warm for 10 minin a 37 °C water bath.
Prepare the probe mixes as required (1:50 volume ratio C2:C1).
Remove the excess water by flicking the slides.
Place the slides in the slide rack and add 200 µL of probe mix to cover each section.
Insert the slide rack into the HybEz oven and incubate for 120 min at 40 °C.
 
During the incubation, prepare 3 L of 1x Wash Buffer (to 2940 mL of milliQ water add 1 bottle (60 mL) of prewarmed (40 °C) 50X RNAscope Wash Buffer) and 5X SSC (to 187.5 mL milliQ water add 62.5 mL 20X SSC).
Wash the slides in a slide holder 2 times for 2 min in wash buffer at Room temperature.
Then store slides 2 min in 5x SSC at Room temperature.
SECTION: Pretreatments
4. Hydrogen Peroxide treatment
Collect the slides from the PBS wash, remove excess PBS and lay slides on a flat surface.
Add enough drops of Hydrogen Peroxide solution to cover the entire tissue section (3--4 drops).
Incubate at Room temperaturefor 10 min.
Remove the solution and immediately wash with milliQ water using the slide holder. Repeat wash 3 times.
5. Target Retrieval
Using forceps briefly transfer the slides in the slide holder into the boling milliQ water before transferring the slides into the boiling Target Retrieval solution. Cover with foil and incubate for 15 min.
 

 

Transfer the slides to a slide holder filled with milliQ water at Room temperature for 5 min Wash the slides in milliQ water 3 more times for 5 min each, shaking from time to time.
Wash the slides for 5 minin 100 % (v/v) ethyl alcohol and allow to completely dry atRoom temperature.
Using the hydrophobic pen, create a barrier around the sections.
6. Protease III treatment
Once the slides have completely dried, put them in the HybEz slide rack and add enough RNAscope Protease III drops to covert the tissue (3-4 drops).
Place the slide rack in the HybEz Humidity Control tray, close the lid and insert into the HybEz oven. 
Incubate at 40 °C for 15 min.
During this time warm the 50x RNAscope Wash buffer in a 37 °C water bath.
At the end of the incubation remove the HybEz Humidity Control tray from the oven. 
Remove the slide rack and replace the tray back into the oven.
Flick each slides to remove the Protease III solution and wash 3 times in milliQ water at Room temperature for 5 min in a slide holder.
SECTION: RNAscope Assay
8. Amp hybridization steps
Amp1
Remove the excess liquid by flicking the slides and add enough Amp1 to cover the tissue.
Insert the slide rack into the HybEz oven and incubate for 30 min at 40 °C.
Wash the slides in a slide holder 2 times for 2 min in wash buffer at Room temperature.
Amp2
Remove the excess liquid by flicking the slides and add enough Amp2 to cover the tissue.
Insert the slide rack into the HybEz oven and incubate for 30 min at 40 °C.
Wash the slides in a slide holder 2 times for 2 min in wash buffer at Room temperature.
Amp3
Remove the excess liquid by flicking the slides and add enough Amp3 to cover the tissue.
Insert the slide rack into the HybEz oven and incubate for 15 min at 40 °C.
Wash the slides in a slide holder 2 times for 2 min in wash buffer at Room temperature.

SECTION: RNAscope Assay
9. Develop HRP-C1 signal
Remove the liquid by flicking the slides and add enough HRP-C1 to cover the tissue.
Insert the slide rack into the HybEz oven and incubate for 15 min at 40 °C.
Wash the slides in a slide holder 2 times for 2 min in wash buffer at Room temperature.
Remove the liquid by flicking the slides and add enough Opal fluorophore (diluted 1:1500 in TCA buffer) to cover the tissue.
Insert the slide rack into the HybEz oven and incubate for 30 min at 40 °C.
Wash the slides in a slide holder 2 times for 2 min in wash buffer at Room temperature.
Remove the liquid by flicking the slides and add enough HRP blocker to cover the tissue.
Insert the slide rack into the HybEz oven and incubate for 15 min at 40 °C.
Wash the slides in a slide holder 2 times for 2 min in wash buffer at Room temperature.
SECTION: RNAscope Assay
10. Develop HRP-C2 signal
Remove the liquid by flicking the slides and add enough HRP-C2 to cover the tissue.
Insert the slide rack into the HybEz oven and incubate for 15 min at 40 °C.
Wash the slides in a slide holder 2 times for 2 min in wash buffer at Room temperature.
Remove the liquid by flicking the slides and add enough Opal fluorophore (diluted 1:1500 in TCA buffer) to cover the tissue.
Insert the slide rack into the HybEz oven and incubate for 30 min at 40 °C.
Wash the slides in a slide holder 2 times for 2 min in wash buffer at Room temperature.
Remove the liquid by flicking the slides and add enough HRP blocker to cover the tissue.
Insert the slide rack into the HybEz oven and incubate for 15 min at 40 °C.
Wash the slides in a slide holder 2 times for 2 min in wash buffer at Room temperature.
SECTION: RNAscope Assay
11. Counterstain and mount
Remove the liquid by flicking the slides and add enough DAPI to cover the tissue.
Incubate for 30 s at Room temperature.
Remove the liquid by flicking the slides and add 1-2 drops Prolong Gold Antifade Montant to each slide and cover with a cover slip. 
Allow to dry for at least 30 min. Store slides in the dark at 4 °C.