RNA in situ hybridization on pancreatic sections using RNAscope® technology

Caroline CT Tremblay, Julien Ghislain, Vincent Poitout

Published: 2022-05-05 DOI: 10.17504/protocols.io.e6nvw9367gmk/v1

Abstract

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

Preparation of cryosections

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 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 8 µm thickness and collect on Superfrost Plus microscopic slides.Store the sections at -80°C until ready for staining.

Pretreatments

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 0h 30m 0s.

Wash the slides in a slide holder with PBS for at least 0h 5m 0s shaking from time to time.

Prepare 1x Target Retrieval solution (1 bottle + 630mL milliQ water) in a 1L 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 700mLof milliQ water in a 1L glass beaker that will be used to prewarm the slides (step 5).

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 0h 10m 0s.

Remove the solution and immediately wash with milliQ water using the slide holder. Repeat wash 3 times.

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 0h 15m 0s.

Note

Do not let the slides cool down in the Target Retrieval solution.

Note

Incubation time for the Target Retrieval step may vary. For mice pancreas incubate for maximum 8 minutes and 30 secondes.

Transfer the slides to a slide holder filled with milliQ water at Room temperature for 0h 5m 0s Wash the slides in milliQ water 3 more times for 0h 5m 0s each, shaking from time to time.Wash the slides for 0h 5m 0sin 100% (v/v) ethyl alcohol and allow to completely dry atRoom temperature.

Using the hydrophobic pen, create a barrier around the sections.

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 0h 15m 0s.

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 0h 5m 0s in a slide holder.

RNAscope Assay

Probe hybridization (2 probe method)

Determine the quantity of probes, transfer to 1.5 ml tubes and warm for 0h 10m 0sin 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 200µL of probe mix to cover each section.

Insert the slide rack into the HybEz oven and incubate for 2h 0m 0s at 40°C.

Note

Do not mix probes of the same channel.

During the incubation, prepare 3L of 1x Wash Buffer (to 2940mL of milliQ water add 1 bottle (60mL) of prewarmed (40°C) 50X RNAscope Wash Buffer) and 5X SSC (to 187.5mL milliQ water add 62.5mL 20X SSC).Wash the slides in a slide holder 2 times for 0h 2m 0s in wash buffer at Room temperature.Then store slides 0h 2m 0s in 5x SSC at Room temperature.

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 0h 30m 0s at 40°C.