May 05, 2022
RNA in situ hybridization on pancreatic sections using RNAscope® technology
- 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, Julien Ghislain, Vincent Poitout 2022. RNA in situ hybridization on pancreatic sections using RNAscope® technology. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvw9367gmk/v1
Manuscript citation:
Croze ML, Flisher MF, Guillaume A, Tremblay C, Noguchi GM, Granziera S, Vivot K, Castillo VC, Campbell SA, Ghislain J, Huising MO, Poitout V. Free fatty acid receptor 4 inhibitory signaling in delta cells regulates islet hormone secretion in mice. Mol Metab. 2021 Mar;45:101166. doi: 10.1016/j.molmet.2021.101166. Epub 2021 Jan 20. PMID: 33484949
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: July 28, 2020
Last Modified: May 05, 2022
Protocol Integer ID: 39786
Keywords: pancreas, islet, RNA in situ hybridization
Funders Acknowledgement:
CIHR
Grant ID: 0406014428
NIH
Grant ID: 5R01DK058096-15
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.
Image Attribution
Caroline Tremblay
Materials
Phosphate Buffered Saline 10x (solution)Bio Basic Inc.Catalog #PD8117
Paraformaldehyde 10% bufferedNewcomer SupplyCatalog #13301A
Sucrose Ultra PureBioshopCatalog #SUC507.5
Superfrost Plus Microscope SlidesFischer ScientificCatalog #12-550-15
OCT (Optimal Cutting Temperature compound)Sakura FinetekCatalog #4583
MilliQ water
100% EthanolContributed by users
ImmEdge hydrophobic barrier pap penVector LaboratoriesCatalog #H-4000
ProLong™ Gold Antifade MountantThermo FisherCatalog #P36930
RNAscope Multiplex Fluorescent Detection Reagents V2Avanced Cell DiagnosticsCatalog #323110
1 Liter SSC [20X] (Sodium Chloride-Sodium Citrate) (0.3M sodium citrate, 3M NaCl, pH7.0)G-BiosciencesCatalog #R019
Opal 620 Reagent PackPerkin ElmerCatalog #FP1495001KT
Opal 520 Reagent PackPerkin ElmerCatalog #FP1487001KT
Opal 690 Reagent PackPerkin ElmerCatalog #FP1497001KT
Opal 570 Reagent PackPerkin ElmerCatalog #FP1488001KT
DAPI (2.5mg/mL)Contributed by users
Equipment
HybEZ II system
NAME
hybridization oven
TYPE
Advanced Cell Diagnostics
BRAND
PN 321710/321720
SKU
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 a chemical hood.
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).
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 Overnight 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 8 µm thickness and collect on Superfrost Plus microscopic slides.
Store the sections at -80 °C until ready for staining.
2h
Pretreatments
Pretreatments
2h
2h
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 00:30:00 .
Wash the slides in a slide holder with PBS for at least 00:05:00 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 mL of milliQ water in a 1 L glass beaker that will be used to prewarm the slides (step 5).
35m
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 temperature for 00:10:00 .
Remove the solution and immediately wash with milliQ water using the slide holder. Repeat wash 3 times.
10m
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 00:15:00 .
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 00:05:00 Wash the slides in milliQ water 3 more times for 00:05:00 each, shaking from time to time.
Wash the slides for 00:05:00 in 100 % (v/v) ethyl alcohol and allow to completely dry atRoom temperature .
Using the hydrophobic pen, create a barrier around the sections.
30m
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 00:15:00 .
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 00:05:00 in a slide holder.
20m
RNAscope Assay
RNAscope Assay
1d
1d
Probe hybridization (2 probe method)
Determine the quantity of probes, transfer to 1.5 ml tubes and warm for 00:10:00 in 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 02:00:00 at 40 °C .
Note
Do not mix probes of the same channel.
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 00:02:00 in wash buffer at Room temperature .
Then store slides Overnight in 5x SSC at Room temperature .
2h 14m
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 00:30:00 at 40 °C .
Wash the slides in a slide holder 2 times for 00:02:00 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 00:30:00 at 40 °C .
Wash the slides in a slide holder 2 times for 00:02:00 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 00:15:00 at 40 °C .
Wash the slides in a slide holder 2 times for 00:02:00 in wash buffer at Room temperature .
1h 21m
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 00:15:00 at 40 °C .
Wash the slides in a slide holder 2 times for 00:02:00 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 00:30:00 at 40 °C .
Wash the slides in a slide holder 2 times for 00:02:00 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 00:15:00 at 40 °C .
Wash the slides in a slide holder 2 times for 00:02:00 in wash buffer at Room temperature .
1h 6m
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 00:15:00 at 40 °C .
Wash the slides in a slide holder 2 times for 00:02:00 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 00:30:00 at 40 °C .
Wash the slides in a slide holder 2 times for 00:02:00 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 00:15:00 at 40 °C .
Wash the slides in a slide holder 2 times for 00:02:00 in wash buffer at Room temperature .
1h 6m
Counterstain and mount
Remove the liquid by flicking the slides and add enough DAPI to cover the tissue.
Incubate for 00:00:30 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 00:30:00 . Store slides in the dark at 4 °C .
30m 30s