Jul 25, 2025

Public workspaceExpanded RNAscope™ In Situ Hybridization Assay 

Applications in Plant Sciences
DOI
dx.doi.org/10.17504/protocols.io.14egn6pp6l5d/v1
  • Lena Hileman1,
  • Brooklyn Anaya1,
  • Austin Nguyen1,
  • Kelly Matsunaga1
  • 1University of Kansas
Brooklyn Anaya
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DOI: https://dx.doi.org/10.17504/protocols.io.14egn6pp6l5d/v1
External link: https://bsapubs.onlinelibrary.wiley.com/doi/10.1002/aps3.70020
Protocol Citation: Lena Hileman, Brooklyn Anaya, Austin Nguyen, Kelly Matsunaga 2025. Expanded RNAscope™ In Situ Hybridization Assay . protocols.io https://dx.doi.org/10.17504/protocols.io.14egn6pp6l5d/v1
Manuscript citation:
Anaya, B. M., A. T. Nguyen, K. K. S. Matsunaga, and L. C. Hileman. 2025. Expanded application to plant reproductive tissues of a branched DNA probe-based in situ hybridization method. Applications in Plant Sciences e70020. https://doi.org/10.1002/aps3.70020
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: October 22, 2024
Last Modified: July 25, 2025
Protocol Integer ID: 110575
Keywords: branched DNA probes, CYCLOIDEA, Histone H4, in situ hybridization, inflorescence, reproductive cone, RNAscope, Z-probe, situ hybridization assay, histone h4 expression in reproductive tissue, rnascope assay, detection of histone h4 expression, expanded rnascope, reproductive tissues of mimulus lewisii, rnascope ish, cycloidea transcription factor, expression of cycloidea transcription factor, quality ish results in reproductive tissue, histone h4 expression, challenging approaches to gene expression, breadth of plant species, situ hybridization, gene expression, plant development, gymnosperm species, assay, mrna, model angiosperm, specific patterns of gene expression, plant species, reproductive tissue, mimulus lewisii
Funders Acknowledgements:
NSF
Grant ID: 2338756
NSF
Grant ID: 2421327
NSF-GRFP
Grant ID: 1940699
NSF-RaMP
Grant ID: 2319820
K-INBRE
Grant ID: #P20 GM103418
NIGMS
Grant ID: P30GM145499
NSF Major Research Instrumentation Award
Grant ID: 2117449
Abstract
Detecting clear tissue- and organ-specific patterns of gene expression is key to understanding the genetic programs that control plant development. In situ hybridization (ISH) of mRNA is one of the clearest, yet most challenging approaches to gene expression assays. Detection of Histone H4 expression in reproductive tissues of Mimulus lewisii, a model angiosperm, was optimized using the RNAscope ISH assay. The optimized protocol was used to detect Histone H4 expression in reproductive tissues of two gymnosperm species, Taxodium distichum and Juniperus virginiana, without further need for species-specific optimization. Additionally, the optimized protocol was used to detect expression of CYCLOIDEA transcription factors in M. lewisii reproductive tissues without further optimization and with results similar to those previously reported. The RNAscope assay can quickly and sensitively generate high-quality ISH results in reproductive tissues across a breadth of plant species. 
Materials
Instruments/Software: 
Materials:
  • Fisherbrand‱ Polypropylene Centrifuge Tubes (05-539-9)
  • Glass Scintillation vials [20 mL] (03-341-73)
  • Stainless Steel General-purpose Forceps
  • DABPRESS 6X7 Rosin Cold Plate (dp-cp67-us)
  • Fisherbrand‱ Tissue Path‱ MACROSETTE‱ Processing/Embedding Cassettes (15-182-706)
  • Bell Jar: Thermo Scientific‱ Nalgene‱ Transparent Polycarbonate Classic Design Desiccator (08-642-7)
  • Vollrath Stainless Steel Beakers [50 mL, 100 mL]
  • Chemglass Life Sciences Beaker, Low Form, Griffin, Pyrex [400 mL]
  • DWK Life Sciences Wheaton‱ 20-Slide Glass Staining Dish (08-812-1A)
  • Reynolds Wrap Aluminum Foil 
  • AccuForge Single Edge Blade Clamshell (AGBL-7033-0000)
  • Epredia‱ Ultra Disposable Microtome Blades (30-538-35), Gold Microtome Blades (D554G50)
  • FisherBrand Glass Dropper [2.5 mL]
  • Peel-a-Way Embedding Molds - Truncated (50-465-348)
  • Fisherbrand‱ Superfrost‱ Plus Microscope Slides (22-037-246), Fisherbrand‱ ProbeOn Plus Microscope Slides (22-230-900)
  • Epredia‱ Richard-Allan Scientific‱ Cover Glass [22 x 50 mm 1.5] (22-050-238)
  • Electron Microscopy Sciences Easy Dip‱ Kit [Slide Staining Jars and Racks] (NC0263028) 

Protocol materials
ReagentCitrisolv (d-Limonene)Fisher ScientificCatalog #22-143-975
ReagentParaplast Plus Tissue Embedding MediumGrayine MedicalCatalog #39502004
ReagentEpredia Signature Series ParaffinFisher ScientificCatalog #22-050-128
ReagentEpredia Tissue Section AdhesiveFisher ScientificCatalog #86-014
ReagentPARA/Gard Paraffin RepellentFisher ScientificCatalog #15-183-15
ReagentSilica Gel DesiccantFisher ScientificCatalog #S684-212
ReagentXylenes (Histological)Fisher ScientificCatalog #X3P-1GAL
ReagentRNAscope® Target Retrieval ReagentsAdvanced Cell DiagnosticsCatalog #322000
ReagentRNAscope Hydrogen Peroxide (H2O2)Advanced Cell DiagnosticsCatalog #322330
ReagentImmEdge Hydrophobic Barrier PenAdvanced Cell DiagnosticsCatalog #310018
ReagentRNAscope Protease Plus ReagentsAdvanced Cell DiagnosticsCatalog #322330
ReagentRNAscope® Wash Buffer ReagentsAdvanced Cell DiagnosticsCatalog #310091
ReagentRNAscope 2.5 HD AMP 1Advanced Cell DiagnosticsCatalog #322311
ReagentRNAscope 2.5 HD AMP 2Advanced Cell DiagnosticsCatalog #322312
ReagentRNAscope 2.5 HD AMP 3 Advanced Cell DiagnosticsCatalog #322313
ReagentRNAscope 2.5 HD AMP 4Advanced Cell DiagnosticsCatalog #322314
ReagentRNAscope 2.5 HD AMP 5 REDAdvanced Cell DiagnosticsCatalog #322361
ReagentRNAscope 2.5 HD AMP 6 REDAdvanced Cell DiagnosticsCatalog #322362
ReagentHematoxylin Solution, Gill No. 1Merck MilliporeSigma (Sigma-Aldrich)Catalog #GHS116-500ML
ReagentRNAscope Fast Red BAdvanced Cell DiagnosticsCatalog #320459
ReagentRNAscope Fast Red AAdvanced Cell DiagnosticsCatalog #320458
ReagentEcomountBiocare MedicalCatalog #EM897L
ReagentVectamountAdvanced Cell DiagnosticsCatalog #321584
ReagentEUKITT Neo Special Mounting MediumElectron Microscopy SciencesCatalog #15320-25
Reagent10X Hank’s Balanced Salt Solution (HBSS)Thermo Fisher ScientificCatalog #14185-052
ReagentMolecular Biology Grade Water, CorningFisher ScientificCatalog #MT46000CM
ReagentPierce 16% Formaldehyde (w/v), Methanol-freeThermo Fisher ScientificCatalog #28908
ReagentSilwet-L77 [VAC-N-STUFF]Phytotech LabsCatalog #S7777
Troubleshooting
Tissue Fixation & Embedding
Preparation


Make appropriate volume (Amount40 mL , more if needed) of 4% Formaldehyde Solution (form.) by adding Reagent10X Hank’s Balanced Salt Solution (HBSS)Thermo Fisher ScientificCatalog #14185-052 , Amount26 mL ReagentMolecular Biology Grade Water, CorningFisher ScientificCatalog #MT46000CM (MG Water), Amount10 mL ReagentPierce 16% Formaldehyde (w/v), Methanol-freeThermo Fisher ScientificCatalog #28908 , and Amount4 µL ReagentSilwet-L77 [VAC-N-STUFF]Phytotech LabsCatalog #S7777 in a 50 mL falcon tube, distribute into the appropriate amount of scintillation (scint.) vials. 
Note
Make 4% form. in the safety hood. Keep on ice for entire procedure.

Molecular Biology Grade water (not DEPC-treated water) is used throughout the protocol to reduce the possibility of RNase contamination.







Toxic
Fill scint. Vials approximately half full with 4% form. 
Dissect the plant material and submerge tissue immediately into 4% form. in scint. vials.
Note
Tissue should be less than approximately 5 mm in any dimension.

Place scint. vials uncapped and packed into ice in the bottom of the bell jar.
Vacuum Infiltration
Using the vacuum pump attached to the bell jar, pull a full vacuum to Pressure-27 inHg , hold for Duration00:01:00 , quickly release vacuum.

1m
Go to until tissue no longer floats.

Cap scint. vials and place on the rocker atTemperature1 °C toTemperature4 °C for Duration12:00:00 to Duration24:00:00 .
Note
Fixation time is critical, optimize to between 12-24 hours. While standard ISH protocols for plant tissues often recommend 12 hr fixation, we found that 24 hr fixation, coupled with 10 min. Protease Plus treatment (see below) worked well.


Overnight
Prep EtOH solutions for dehydration steps and place in Temperature4 °C for the following day.

Dehydration
Ethanol Series (Formaldehyde solution to EtOH)
Dehydrate the samples through the following EtOH series on the rocker at Temperature1 °C to Temperature4 °C , keeping samples in scint. vials:
Note
All EtOH solutions should be pre-chilled at 1-4°C and made from fresh 200 proof EtOH with MG water.

  1. 50% EtOH forDuration01:30:00
  2. 70% EtOH forDuration01:15:00
  3. 85% EtOH forDuration02:15:00
  4. 95% EtOH for Duration01:00:00
  5. 100% EtOH for Duration01:30:00
  6. 100% EtOH for DurationOvernight at Temperature4 °C .

7h 30m
Overnight
Wax Infiltration
Citrisolv Series (EtOH to Wax)
Ensure the vacuum oven is set to Temperature60 °C

Move the rocker to TemperatureRoom temperature

Put samples though the following ReagentCitrisolv (d-Limonene)Fisher ScientificCatalog #22-143-975 series on the rocker at TemperatureRoom temperature , keeping samples in scint. vials: 

1. 100% EtOH for Duration01:00:00
2. 50:50 EtOH:Citrisolv for  Duration02:00:00
Note
Equal parts 100% EtOH and 100% Citrisolv

3. 100% Citrisolv for Duration02:00:00
Note
Remove as much 50:50 EtOH:Citrisolv as possible with pipette before adding 100% Citrisolv.

4. 50:50 Citrisolv:Wax Chips Temperature60 °C DurationOvernight (oven instructions below)
Note
Fill glass staining dish ½ with Citrisolv
Place tissue cassette(s) open and submerged in Citrisolv.

Move tissue into cassette(s): 
Note
Discard most of Citrisolv from scint. vials, but keeping tissue submerged. Quickly dump tissue with remaining Citrisov from scint. vial into open, submerged cassettes in the glass staining dish. Use forceps to take hold of the cassette(s) and shake while submerged in Citrisolv to release bubbles. 

Note
Multiple tissues can be co-processed in single cassettes, but do not over-crowd tissue. 

5h
Bring the glass staining dishes that are half-full with Citrisolv to completely full with ReagentParaplast Plus Tissue Embedding MediumGrayine MedicalCatalog #39502004 .
Safety information
Paraplast Plus contains DMSO for higher efficiency infiltration. Handle with caution.


Toxic
Allow the samples to infiltrate with Citrisolv:wax at Temperature60 °C , DurationOvernight , under Pressure-20 inHg vacuum.
Note
Once the vacuum reaches target pressure, close the oven vacuum valve and turn off vacuum pump.

Note
Put two additional glass staining dishes filled with molten wax (or wax chips) in the oven at this time so these dishes are ready to use the following morning for wax changes.


Overnight
Make 3 changes into fresh molten wax per day for the next 2 days. All changes at Temperature60 °C , under vacuum, as described above.
Note
Approximately 9:00 am, 1:00 pm, 4:30 pm.


Note
With the forceps, quickly transfer the cassette(s) from the current glass dish to the fresh dish. Shake to release bubbles. 
Do not reuse the Citrisolv:wax glass dish for the 100% wax steps.

Overnight
Embedding
For embedding, use DMSO-free ReagentEpredia Signature Series ParaffinFisher ScientificCatalog #22-050-128 .

Move the glass staining dish of molten wax containing cassette(s) from the vacuum oven to the heating compartment of the Epredia HistoStar embedding station.
Embed tissue in the molds. Alternating between heat and cold to orient and solidify the tissue in the bottom of molds as desired.
Once opaque, move the mold to the cold block to set for Duration02:00:00 .
Note
Once fully solidified, either store molds at 4C or proceed to Tissue Sectioning.


2h
Pause
Tissue Sectioning & Slide Mounting
Sectioning
~30 minutes prior to sectioning:
Pre-heat the warming plate to Temperature40 °C and coat “Superfrost Plus” microscope slides with a drop of 100%ReagentEpredia Tissue Section AdhesiveFisher ScientificCatalog #86-014 . Spread over the slide surface and remove excess with a gloved finger (see video), place slides on the warmer to dry. 
Note
 Do not begin mounting until the adhesive has dried. Approximately ten minutes on the warming plate until slides are dry.



Using a razor blade, trim the wax molds into a trapezoidal shape down to the bevel or depth of embedded tissue. Mount the wax block to a microtome mounting block and set the microtome to section at Thikness7 µm .

With a kimwipe, wipe the blade and blade holder with ReagentPARA/Gard Paraffin RepellentFisher ScientificCatalog #15-183-15 , wait a few seconds and then wipe the blade and blade holder with water.
Note
The Paraguard will melt away any wax and prevent adherence of wax slices onto the blade holder, allowing the ribbon to form.
The water will prevent static, helping to keep the ribbons from wrinkling. Blade holder should ideally be damp to allow ribbon to glide over smoothly.

Note
Recommended: section entire volume of embedded tissue laying out ribbons onto static-free, clean surface; then spot check sections for “good morphology”; only mount sections with target morphology onto coated slides for ISH.


Wet-Mounting Sections to Slides
With coated slides and a beaker of MG water on the warmer at Temperature40 °C , use a disposable glass dropper to place 4 drops of MG water evenly spaced from top to bottom of the slide.
Note
~1 cm of space between each drop, or your sections will be too crowded and make later assay steps difficult (i.e., creating hydrophobic barriers).


Leave the slide with water on the warming plate for ~Duration00:00:10 before adding the sections.

10s
Move single sections onto single drops of water on the slide by gently laying the slice onto the pool of water.
Note
Leave section on the water bubble for ~Duration00:02:00 . This allows the water to “stretch” out the wax section and remove wrinkles.
Ideally, your wax sections should be large enough that the corners adhere to the glass slide, leaving the middle on top of the bubble (see image).

With the tissue sections bubbled on top of the water droplets, slowly tilt and tap the droplets out from under the sections onto a paper towel, leaving the sections behind on the slide surface. Flick to remove excess water from under sections. Replace slide back onto the warmer


Leave slides on warmer for ~Duration00:02:00 , flick again to remove excess water.

2m
Once all desired sections are mounted to slides, close the warmer and leave slides DurationOvernight .

Overnight
The following day, move slides from the warmer into a slide box with a layer of ReagentSilica Gel DesiccantFisher ScientificCatalog #S684-212 beads inside unless moving directly to ISH protocol.
Note
Optional stopping point (1). The slides can be preserved in desiccant for 3 months maximum at TemperatureRoom temperature before moving to RNAscope ISH assay.



Pause
RNAscope 2.5 HD Detection Kit (RED) ISH Assay
Part 1: Prepare and Pretreat Samples

3h 30m
Prep:
Turn on oven to Temperature60 °C . 
Move hot plate into fume hood.
Open new bottle of 200 proof EtOH for specific use during ISH protocol. 
Note
Note: each EasyDip slide container used throughout ISH protocol will hold ~100 mL of liquid. Each EasyDip slide rack holds 12 slides.
EasyDip container and slide rack hereafter referred to as “jars” and “racks”

Bake Slides: Place slides on a flat, clean surface (e.g. tray) and bake in a dry oven for Duration01:00:00 at Temperature60 °C .
After, reduce oven to Temperature40 °C .
Note
Optional stopping point (2). Use sectioned tissue within 1 week. Store sections with desiccants at TemperatureRoom temperature .


1h
Pause
During "Bake Slides" step, prepare HybEZ oven: Set HybEZ oven to Temperature40 °C and warm humidity control tray containing paper towel (wetted with DI water) for Duration00:30:00 before use. (Keep tray warm during assay).

30m
Deparaffinize FFPE sections (in fume hood):
1. Load slides into rack and place into a jar with ReagentXylenes (Histological)Fisher ScientificCatalog #X3P-1GAL 2 x Duration00:05:00 , agitate gently.
2. Wash slides in 100% EtOH 2 x Duration00:01:00 .
3. Remove slides from rack. Air dry slides for Duration00:10:00 at TemperatureRoom temperature (or until completely dry).

16m
At the start of the 10 minute drying period: Prepare Amount300 mL fresh 1X Target Retrieval Solution in a 400 mL beaker. Do this by adding Amount30 mL ReagentRNAscope® Target Retrieval ReagentsAdvanced Cell DiagnosticsCatalog #322000 to Amount270 mL MG water.
Cover with foil, bring to a mild boil (see video), and maintain. 
Note
Do not boil more than Duration00:30:00 before use.



Note
Optional stopping point (3). Air dry overnight at TemperatureRoom temperature (must use within Duration24:00:00 ) or proceed directly to the next step.


Pause
Apply hydrogen peroxide:
 
Note
Recommended to start the hydrogen peroxide incubation 10-15 minutes after placing Target Retrieval Solution on hot plate.

  1. Place slides back on flat, clean, dark-colored surface (e.g., tray lined with foil so you can see tissue on slides) and add 2-3 drops (or full coverage) of ReagentRNAscope Hydrogen Peroxide (H2O2)Advanced Cell DiagnosticsCatalog #322330 to each deparaffinized section for Duration00:10:00 at TemperatureRoom temperature .
  2. Remove hydrogen peroxide from the slide by tapping the side onto absorbent paper (i.e., small stack of paper towels).
  3. Place slides into rack, rinse in MG water; dunk 3-5x.
  4. Repeat wash with fresh MG water.



10m
Perform RNAscope Target Retrieval:
  1. Very slowly, submerge the slide rack into the simmering 1X Target Retrieval Solution for Duration00:05:00 .
  2. Immediately transfer hot slide rack to a jar containing MG water. 
  3. Wash slides in MG water; dunk 3-5 times.
  4. Repeat wash with fresh MG water.
  5. Wash slides in 100% EtOH 1x for Duration00:03:00 , agitate gently. 
  6. Remove slides from rack and air dry Duration00:10:00 at TemperatureRoom temperature .
 


18m
Create Hydrophobic Barrier: Place slides back on a flat, clean, dark-colored surface draw around tissue 2-4x using ReagentImmEdge Hydrophobic Barrier PenAdvanced Cell DiagnosticsCatalog #310018 (see image). Let dry completely (~Duration00:10:00 ) at TemperatureRoom temperature .

Note
Shake hydrophobic barrier pen well before use, press tip to a glass surface (e.g., spare slide) to dispense fluid prior to use on sample slides.

10m
Apply Protease Plus:
  1. Place slides into HybEZ Slide rack and lock; add 1-2 drops ReagentRNAscope Protease Plus ReagentsAdvanced Cell DiagnosticsCatalog #322330 to each section resulting in full coverage.
  2. Place HybEZ slide rack in the prewarmed HybEZ Humidity Control Tray with lid and insert back into HybEZ oven. Incubate for Duration00:10:00 at Temperature40 °C .
  3. Remove slides and gently tap on absorbent paper to remove excess liquid.
  4. Load slides into rack.
  5. Wash slides in MG water; dunk 3-5x.
  6. Repeat wash with fresh MG water.

Note
When first putting slides in to incubate, move probes to Temperature40 °C  oven for ~Duration00:10:00 , then let probes come to TemperatureRoom temperature .



10m
Part 2: RNAscope 2.5 Assay
5h
Hybridize Probe:
  1. Remove excess liquid from slides by flicking aggressively (see video), place in the HybEZ Slide rack and lock, add ~4 drops of probe to each section (fully cover).
Note
If, at any time, probe solution spills onto another section, dunk slide into Wash Buffer immediately to prevent any unwanted reactions

  1. Insert the covered tray containing slide rack back into the HybEZ oven forDuration02:00:00 at Temperature40 °C .
Note
Prep materials during this time:
1st hour:
1. Put the bottle of RNAscope 50X ReagentRNAscope® Wash Buffer ReagentsAdvanced Cell DiagnosticsCatalog #310091 in the Temperature40 °C oven for Duration01:00:00 .
2. Make 0.02% Ammonia water (bluing reagent). If making from scratch, begin preparation now; prepare in fume hood. Make this by combining Amount1.43 mL 1N Ammonium Hydroxide and Amount250 mL DI water.
2nd hour:
Remove the 50X Wash Buffer from the oven, reset the oven to Temperature60 °C
1. Prepare Amount1.5 L of 1X Wash Buffer by adding Amount30 mL 50X Wash Buffer to Amount1.47 L DI water.
2. Move Amps 1-6 to the bench, let come to TemperatureRoom temperature (if light sensitive, leave in insulated box).


  1. Remove slides from the HybEZ tray and rack, tap off excess liquid from slides onto absorbent paper.
  2. Wash slides in 1X Wash Buffer for Duration00:02:00 at TemperatureRoom temperature , agitate gently.
  3. Repeat wash with fresh 1X Wash Buffer.
2h 2m
Amp 1 Hybridization:  
  1. Remove excess liquid from slides, place in HybEZ slide rack and lock, add ~2 drops of ReagentRNAscope 2.5 HD AMP 1Advanced Cell DiagnosticsCatalog #322311 to each section (full coverage). 
  2. Insert the closed tray containing slide rack back into the HybEZ oven forDuration00:30:00 atTemperature40 °C .
  3. Remove slides from rack and tap off excess liquid.
  4. Wash slides in 1X Wash Buffer for Duration00:02:00 at TemperatureRoom temperature , agitate gently. 
  5. Repeat wash with fresh 1X Wash Buffer.

32m
Amp 2 Hybridization:
  1. Remove excess liquid from slides, place in HybEZ slide rack and lock, add ~4 drops of ReagentRNAscope 2.5 HD AMP 2Advanced Cell DiagnosticsCatalog #322312 to each section. 
  2. Insert the covered tray containing slide rack back into the HybEZ oven for Duration00:15:00 at Temperature40 °C .
  3. Remove slides from rack and tap off excess liquid.
  4. Wash slides in 1X Wash Buffer for Duration00:02:00 at TemperatureRoom temperature , agitate gently.
  5. Repeat wash with fresh 1X Wash Buffer.

17m
Amp 3 Hybridization:
  1. Remove excess liquid from slides, place in HybEZ slide rack and lock, add ~4 drops of ReagentRNAscope 2.5 HD AMP 3 Advanced Cell DiagnosticsCatalog #322313 to each section. 
  2. Insert the covered tray containing slide rack back into the HybEZ oven for Duration00:30:00 at Temperature40 °C .
  3. Remove slides from rack and tap off excess liquid.
  4. Wash slides in 1X Wash Buffer for Duration00:02:00 at TemperatureRoom temperature , agitate gently. 
  5. Repeat wash with fresh 1X Wash Buffer.

32m
Amp 4 Hybridization:
  1. Remove excess liquid from slides, place in HybEZ slide rack and lock, add ~4 drops of ReagentRNAscope 2.5 HD AMP 4Advanced Cell DiagnosticsCatalog #322314 to each section. 
  2. Insert the covered tray containing slide rack back into the HybEZ oven for Duration00:15:00 at Temperature40 °C .
  3. Remove slides from rack and tap off excess liquid.
  4. Wash slides in 1X Wash Buffer for Duration00:02:00 ta@t TemperatureRoom temperature , agitate gently. 
  5. Repeat wash with fresh 1X Wash Buffer.
Note
Turn OFF HybEZ oven at this time. Following incubations are at TemperatureRoom temperature .



17m
Amp 5 Hybridization:
  1. Remove excess liquid from slides, place in HybEZ slide rack and lock, add ~4 drops of ReagentRNAscope 2.5 HD AMP 5 REDAdvanced Cell DiagnosticsCatalog #322361 to each section. 
  2. Incubate the covered tray containing the HybEZ slide rack for Duration01:00:00 at TemperatureRoom temperature .
  3. Remove slides from rack and tap off excess liquid.
  4. Wash slides in 1X Wash Buffer for Duration00:02:00 at TemperatureRoom temperature , agitate gently. 
  5. Repeat wash with fresh 1X Wash Buffer.

1h 2m
Amp 6 Hybridization:
  1. Remove excess liquid from slides, place in HybEZ slide rack and lock, add ~4 drops of ReagentRNAscope 2.5 HD AMP 6 REDAdvanced Cell DiagnosticsCatalog #322362 to each section. 
  2. Incubate the covered tray containing the HybEZ slide rack forDuration00:15:00 at TemperatureRoom temperature .
  3. Remove slides from rack and tap off excess liquid.
  4. Wash slides in 1X Wash Buffer for Duration00:02:00 at TemperatureRoom temperature , agitate gently. 
  5. Repeat wash with fresh 1X Wash Buffer.


17m
Detect Signal:
Briefly spin down ReagentRNAscope Fast Red BAdvanced Cell DiagnosticsCatalog #320459 and pipette mix 1 volume of RED-B to 60 volumes of ReagentRNAscope Fast Red AAdvanced Cell DiagnosticsCatalog #320458 .
Note
Do NOT prep ahead of time: must use within 3-5 minutes.

Note
Be aware of the light sensitivity of the reagents.

RED-A is stored in a dropper tube. Drop solution into an epi tube to approximate amount needed.
(Amount150 µL total is enough to cover 12 sections).

  1. Remove excess liquid from slides, place in the HybEZ slide rack and lock, pipette RED solution onto tissue section to cover.
  2. Incubate covered tray containing HybEZ slide rack for Duration00:10:00 at TemperatureRoom temperature .
  3. Remove slides and tap off excess liquid onto absorbent paper.
  4. Wash slides in TAP water, dunk 3-5x
Note
Prepare 50% Hematoxylin counterstain at this time. Add Amount50 mL ReagentHematoxylin Solution, Gill No. 1Merck MilliporeSigma (Sigma-Aldrich)Catalog #GHS116-500ML to Amount50 mL DI water.




10m
 At this time the expression of the probes can be visualized under the microscope. Observe the slides before deciding whether or not to continue to the counterstain.
Counterstain:
  1. Place slides in 50% Hematoxylin I for Duration00:02:00 at TemperatureRoom temperature . wash in TAP water, dunk 3-5x, repeat with fresh tap water.
  2. Wash slides for Duration00:00:10 in 0.02% Ammonia water, followed by dunking 3-5x in TAP water.
Expected result
Hematoxylin should render slides purple. Ammonia water should render slides blue.

2m 10s
Mount Slides:
  1. Place slides on a flat, clean surface and dry slides in Temperature60 °C dry oven for Duration00:15:00 .
  2. Dip slides into fresh pure (not recycled) xylene, dunk 5-6x, to remove the hydrophobic barrier. 
  3. Immediately apply mounting media (either ReagentEcomountBiocare MedicalCatalog #EM897L , ReagentVectamountAdvanced Cell DiagnosticsCatalog #321584 , or ReagentEUKITT Neo Special Mounting MediumElectron Microscopy SciencesCatalog #15320-25 ) directly onto the sections and slide (~4 drops total) before the xylene dries. Place a coverslip over the section.
  4. Air dry until cover slip is set (Duration00:05:00 or DurationOvernight ).
Note
RED substrate is alcohol sensitive. Do not dehydrate in alcohol!




25m
Imaging
Image on a compound microscope without filters (white light) equipped with digital camera and image capture software.
Note
For example, Nikon Eclipse scope with DS-Fi3 camera and NIS Elements D software.

Protocol references
Bowling, A. J., Pence, H. E., & Church, J. B. (2014). Application of a novel and automated branched DNA in situ hybridization method for the rapid and sensitive localization of mRNA molecules in plant tissues. Applications in Plant Sciences, 2(4), 1400011. https://doi.org/10.3732/apps.1400011

Jackson, D. P. 1992. In-situ hybridisation in plants. In S. J. Gurr, M. J. McPherson, and D. J. Bowles [eds.], Oxford University Press.