Feb 19, 2025

Public workspaceMultiplexed RNA FISH on Expanded Mouse Brain Slices V.1

DOI
https://dx.doi.org/10.17504/protocols.io.dm6gpzj28lzp/v1
Multiplexed RNA FISH on Expanded Mouse Brain Slices
  • Rachel Flannery1,
  • Molly Logsdon1,
  • Laura Roy1,
  • Kevin Cao1,
  • Jayaram Chandrashekar1
  • 1Allen Institute for Neural Dynamics
  • Allen Institute for Neural Dynamics
Hannah Belski
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DOI: https://dx.doi.org/10.17504/protocols.io.dm6gpzj28lzp/v1
Protocol Citation: Rachel Flannery, Molly Logsdon, Laura Roy, Kevin Cao, Jayaram Chandrashekar 2025. Multiplexed RNA FISH on Expanded Mouse Brain Slices. protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gpzj28lzp/v1
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: May 17, 2024
Last Modified: February 19, 2025
Protocol Integer ID: 99985
Keywords: In Situ Hybridization, FISH, Tissue Slice, Tissue Clearing, Hydrogel, RNase Free, Tissue Expansion, Spatial Transcriptomics, multiplexed rna fish, expanded mouse brain slices recent advance, high resolution imaging of cell morphology, single cell rna sequencing, thick slices of brain tissue, spatial transcriptomic, expansion microscopy, brain tissue, retrospective fish, multiscale anatomy, cell morphology, multiple rounds of multiplexed fluorescent, brain slice, multiplexed fluorescent, field of neuroscience, high resolution imaging, neuroscience, rna, tissue clearing, cell, robust trex gel chemistry, situ hybridization, tissue, mfish
Abstract
Recent advances in single cell RNA sequencing, taxonomic classifications of cell types, and high resolution imaging of cell morphology has primed the field of neuroscience for new discoveries. Spatial transcriptomics has the potential to bridge these data modalities to each other by linking the molecular identity of cells to their native biological context.

This protocol describes a method that utilizes expansion microscopy to perform multiple rounds of multiplexed fluorescent in situ hybridization (mFISH) on thick slices of brain tissue. It is adapted from EASI-FISH, where we have incorporated the more robust TREx gel chemistry to achieve better sample durability for ease of sample handling and extended rounds of probing. The protocol is optimized for retrospective FISH on fixed tissue using modalities such as optical physiology and tissue clearing/multiscale anatomy.

Materials

Note
All solutions and equipment must be kept RNase free to preserve RNA quality. Prepare solutions in a PCR hood that has been sprayed thoroughly with RNAse Away™. Wear a lab coat, mask, and gloves to prevent contamination. Spray gloves thoroughly with RNAse Away™ and dry with a Kimwipe® regularly. Milli-Q water can be substituted for nuclease free water.

ReagentRNase AWAY™ Spray Bottle, RNase in spray bottle; 475mLThermo FisherCatalog #7002PK
ReagentPBS - Phosphate-Buffered Saline (10X) pH 7.4Thermo Fisher ScientificCatalog #AM9625
ReagentN,N′-MethylenebisacrylamideMerck MilliporeSigma (Sigma-Aldrich)Catalog #M7279
ReagentAcrylamideMerck MilliporeSigma (Sigma-Aldrich)Catalog #A9099
ReagentSodium chloride solutionMerck MilliporeSigma (Sigma-Aldrich)Catalog #S6546
ReagentUltraPure 0.5M EDTA pH 8.0Invitrogen - Thermo FisherCatalog #15575020
Reagent10% SDS solutionThermo Fisher ScientificCatalog #15553027
ReagentTriton X-100Merck MilliporeSigma (Sigma-Aldrich)Catalog #T8787-50ML
ReagentTris-HCl, 1M Solution, pH 8.0, Molecular Biology Grade, Ultrapure, Thermo Scientific ChemicalsThermo Fisher ScientificCatalog #AAJ22638K2
Reagent5M Sodium Chloride, 1000mlPromegaCatalog #V4221
Reagent10% SDS solutionThermo Fisher ScientificCatalog #15553027
ReagentProteinase K, Molecular Biology Grade - 2 mlNew England BiolabsCatalog #P8107S
ReagentHCR Probe Hybridization BufferMolecular Instruments
ReagentHCR Probe Wash BufferMolecular Instruments
ReagentHCR Amplification BufferMolecular Instruments
ReagentDNase I Reaction Buffer - 6.0 mlNew England BiolabsCatalog #B0303S
ReagentDNase I (RNase-free) - 5,000 unitsNew England BiolabsCatalog #M0303L

Equipment
22x22 mm Square Cover Glass
NAME
Corning
BRAND
2855-22
SKU
https://ecatalog.corning.com/life-sciences/b2b/US/en/Glassware/Cover-Glass/Corning%C2%AE-Square-%232-Cover-Glass/p/2855-22
LINK
Product Number: 2855-22; Qty./Pk: 100 / Pk; Qty./Cs: 1000 / Cs; Size: 22x22 mm (approx); Sterile: No; Thickness: 0.17 - 0.25 mm
SPECIFICATIONS

Equipment
Synthetic Sable Brushes, #3
NAME
Ted Pella
BRAND
11816
SKU
https://www.tedpella.com/brush_html/brush.aspx#_11806
LINK
Synthetic Sable Brushes, #3, 2.0mm W x 13.0mm L
SPECIFICATIONS

Equipment
Electron Microscopy Sciences SuperFrost™ End Slide End Slide 1 mm thick
NAME
Electron Microscopy Sciences 7186701
BRAND
50-949-343
SKU
https://www.fishersci.com/shop/products/end-slide-1-mm-thick-1gs-1/50949343?searchHijack=true&searchTerm=50-949-343&searchType=RAPID&matchedCatNo=50-949-343
LINK

Equipment
Sterile Microcentrifuge Tubes with Screw Caps
NAME
Fisherbrand
BRAND
02-681-374
SKU
https://www.fishersci.com/shop/products/fisherbrand-sterile-microcentrifuge-tubes-screw-caps-6/02681374
LINK
Max. RCF 18,000 x g Autoclavable Autoclavable Color Natural Includes O-ring Closure Type Screw Cap Closure Material Polypropylene Capacity (Metric) 2 mL Material Polypropylene Sterility Sterile Bottom Shape Skirted
SPECIFICATIONS

Equipment
AirClean Systems AC600 Series UPUV Class 1 Biological Safety Workstation
NAME
PCR hood
TYPE
Midwest Production Supply
BRAND
AC632UPUV
SKU
https://aircleansystems.com/products/combination-pcr-workstation
LINK
No ductwork required; No installation required – plugs directly into a standard 110V or 220V electrical outlet; Clear polycarbonate shell for 360° visibility; UVTect® microprocessor controller UV and fluorescent lights; Seamless plastic design -no joints or gaps in construction; Lab event timer; Digital UV light timer :01-59 minutes; Access port for electrical cords; Weight: 163 lbs; External Size: 32"Wx24"Dx30"H or 48"Wx24"Dx32"H
SPECIFICATIONS

Equipment
SimpliAmp™ Thermal Cycler
NAME
Thermal Cycler
TYPE
Applied Biosystems™
BRAND
A24811
SKU
https://www.thermofisher.com/order/catalog/product/A24811
LINK
Thermal Accuracy ±0.25°C (35°C to 99.9°C); High-throughput Compatibility Not High-throughput Compatible (Manual); For Use With (Equipment) GeneAmp 2700, GeneAmp 9700, Veriti Thermal Cycler, 2720 Thermal Cycler; Thermal Uniformity <0.5°C (20 sec. after reaching 95°C); Dimensions 18.1 x 9.5 x 8.3 in. (46 x 24 x 21 cm); Capacity 96-well, 3 Zones VeriFlex; Voltage 100/240 V; Display Type 8 in. Color TFT LCD Touchscreen; Product Type Thermal Cycler; Warranty Limited Warranty; Thermal Range 0°C to 100°C; User Interface USB, Wi-Fi, Ethernet; Wattage 600 W; Max. Ramp Rate 4°C/sec. (Block), 3°C/sec. (Sample); No. of Programs >1000; Weight 18.3 lb. (8.3 kg); Block Configurations 96-well, 0.2 mL VeriFlex Block
SPECIFICATIONS
RECIPES

4.04M Sodium Acrylate:
Safety information
  • Acrylic acid is toxic if swallowed, inhaled, or absorbed through the skin. It is flammable, corrosive, and an environmental hazard. Handle only in a fume hood while wearing a mask, chemically resistant gloves, protective clothing, and eye protection. Keep away from heat, sparks, open flames, and hot surfaces. Dispose of acrylic acid and any contaminated consumables in a hazardous waste stream.

AB
14.6M Acrylic Acid13.75 mL
Nuclease Free Water11.25 mL + extra to reach 50 mL
10N NaOH18 mL
1N NaOH2.5 mL to 5 mL

50% Acrylamide:
Safety information
  • Acrylamide powders and solutions are toxic if swallowed, inhaled, or absorbed through the skin. It is a mutagen, teratogen and a carcinogen. Handle only in a fume hood while wearing a mask, chemically resistant gloves, protective clothing, and eye protection. Dispose of acrylamide and any contaminated consumables in a hazardous waste stream.
  • Combine the following reagents and vortex to combine at TemperatureRoom temperature until fully dissolved. Store at Temperature-20 °C .
ReagentsAmount
Acrylamide5.0 g
Nuclease Free Water10 mL
Total10 mL

2% N,N-Methylenebisacrylamide (N,N-Mba):
Safety information
  • N,N-Methylenebisacrylamide (N,N-Mba) is toxic if swallowed, inhaled, or absorbed through the skin. It is a mutagen, carcinogen, and teratogen. Handle only in a fume hood while wearing a mask, chemically resistant gloves, protective clothing, and eye protection. Dispose of N, N - Mba and any contaminated consumables in a hazardous waste stream.
  • Combine the following reagents and vortex to combine at TemperatureRoom temperature until fully dissolved. Store at Temperature-20 °C .
ReagentsAmount
N,N-Methylenebisacrylamide0.2g
Nuclease Free Water10 mL
Total10 mL

10% VA-044:*
  • Add reagents and mix to combine until solution is clear. Aliquot into 500 µL aliquots and store at Temperature-20 °C .
ReagentsAmount
VA-0440.1 g
Nuclease Free Water10 mL

TREx Gel Solution: 1.1 M Sodium Acrylate, 2.0M Acrylamide, 0.32 mM N,N-Mba, 1X PBS in water (9.4 mL)
  • Combine the following reagents and invert the tube several times to combine. 10% VA-044 will activate the TREx gel solution and allow it to polymerize. Do not add 10% VA-044 until right before applying to sample. Store at Temperature-20 °C .

Safety information
  • Acrylamide powders and solutions are toxic if swallowed, inhaled, or absorbed through the skin. It is a mutagen, teratogen and a carcinogen. Handle only in a biosafety fume hood while wearing a mask, chemically resistant gloves, protective clothing, and eye protection. Dispose of acrylamide and any contaminated consumables in a hazardous waste stream.

  • Sodium acrylate is an environmental toxin. Handle only while wearing a mask, gloves, and protective clothing. Dispose of sodium acrylate and any contaminated consumables in a hazardous waste stream.

ReagentsAmount
Sodium Acrylate2.7 mL
50% Acrylamide2.5 mL
2% N-N Methylenebisacrylamide24.6 µL
10x PBS1 mL
Nuclease Free Water3.4 mL
10% VA-044*110 µL
Total9.4 mL
* Do not add until right before applying to sample

50 mM ProK Buffer - 0.5% TritonX, 50 mM Tris-HCl-pH8, 50 mM NaCl, 1mM EDTA, 0.3% SDS in water (100 mL):
  • Add ingredients to a glass bottle on a stir plate, starting with some of the Nuclease Free Water to aid in dissolving. Stir until the solution is clear or DurationOvernight .
  • Store at TemperatureRoom temperature .
ReagentsAmount
10% Triton X-100*5 mL
1M Tris Base pH 85 mL
5M Sodium Chloride1 mL
0.5M EDTA200 µL
10% SDS2 mL
Water85.8
Total100 mL
*Note: Triton X-100 is highly viscous

Proteinase K Solution (ProK Solution): 0.01% ProK in ProK Buffer
  • Make the solution in each tube containing a gelled sample. Begin by adding 50 mM ProK Buffer to the tube, then add Proteinase K (ProK). Invert tube several times to gently mix.
  • Keep ProK TemperatureOn ice .

ReagentsAmount
50 mM ProK Buffer742.5 µL
ProK (800 U/mL)7.5 µL
Total750 µL

FISH Probe Solution: 50 μM Rn28s, 10 nM Probes in Hybridization Buffer (300 μL):
  • Add 1 µM Rn28s Solution and Probes to Hybridization Buffer. Invert tube several times to combine. Use 300 µL probe solution per sample. If using fewer probes, replace lost volume with hybridization buffer.
Safety information
  • Hybridization buffer contains 10-30% formamide. Formamide is toxic, carcinogenic, and teratogenic. Handle probe wash buffer in a well-ventilated area. Wear chemical resistant gloves, a mask, and protective clothing when handling. Dispose of hybridization buffer and any contaminated consumables in a hazardous waste stream.

ReagentsAmount
1 µM Rn28s Solution15 µL
Probe 13 µL
Probe 23 µL
Probe 33 µL
Hybridization Buffer282 µL
Total300 µL

Hairpin Solution: 30 nM Hairpins
  • Add hairpins to amplification buffer, taking care not to cross contaminate pipette tips. If less hairpin is used, replace lost volume with amplification buffer. Similarly, if more hairpins are being used, remove amplification buffer to ensure total volume per sample remains 200 µL

Note
  • The hairpin initiator sequence should match the corresponding probe initiator sequence. However, only one hairpin initiator sequence should match the probe.

  • For example: Gad1 - B1 probe needs to be paired with the B1 - 488 hairpin to be viewed in the 488 channel. No other B1 hairpins should be applied.

ReagentsAmount
Hairpin 1 H12 µL
Hairpin 1 H22 µL
Hairpin 2 H12 µL
Hairpin 2 H22 µL
Hairpin 3 H12 µL
Hairpin 3 H22 µL
Amplification Buffer188 μL
Total200 μL

5x SSCT - 5x SSC, 0.2% Tween 20 (50 mL):
  • Add 10% Tween 20 to 20x SSC and nuclease-free water. Vortex until combined. Store at TemperatureRoom temperature .

ReagentsAmount
20x SSC12.5 mL
10% Tween 201 mL
Nuclease Free Water37 mL
Total50 mL

0.5x SSCT - 0.5x SSC, 0.2% Tween 20 (50 mL):
  • Add 10% Tween 20 to 20x SSC and nuclease-free water. Vortex until combined. Store at TemperatureRoom temperature .
ReagentsAmount
20x SSC1.25 mL
10% Tween 201 mL
Nuclease Free Water47.75 mL
Total50 mL

DNase Buffer Solution (750 μL):
  • Thaw DNase Buffer at room temp. Vortex until combined.
AB
10x DNase buffer75 μL
Water675 μL
Total Volume750 μL

DNase Solution - 1X DNase Buffer, (500 μL):
  • Thaw DNase Buffer at room temperature. Keep DNAse I on ice. Combine water and DNase Buffer and add to sample tubes. Add DNase I directly to sample tubes. Rock gently to combine.
AB
DNase I*20 μL
10x DNase buffer 50 μL
Water430 μL
Total Volume500 μL
Note: Keep DNase I on ice





Troubleshooting
Safety warnings
Formamide:
Hybridization buffer and probe wash buffer contains 10-30% formamide by weight. Formamide is toxic, carcinogenic, and teratogenic. Wear gloves, a mask, and protective clothing when handling. Dispose of formamide and any contaminated consumables in a hazardous waste stream. Store probe wash buffer and hybridization buffer at Temperature-80 °C .

Acrylamide:
Acrylamide powders and solutions are toxic if swallowed, inhaled, or absorbed through the skin. It is a mutagen, teratogen, and a carcinogen. Handle solid acrylamide in a chemical fume hood while wearing a mask, chemically resistant gloves (glove specs), protective clothing, and eye protection. Dispose of acrylamide and any contaminated consumables in a hazardous waste stream. Store aqueous solutions at Temperature-20 °C ; solid acrylamide can be stored at TemperatureRoom temperature .

Sodium Acrylate:
Sodium acrylate is an environmental toxin. Handle only while wearing a mask, gloves, and protective clothing.
Dispose of sodium acrylate and any contaminated consumables in a hazardous waste stream. Store sodium acrylate at Temperature-20 °C .

N,N-Methylenebisacrylamide:
N,N-Methylenebisacrylamide (N,N-Mba) is toxic if swallowed, inhaled, or absorbed through the skin. It is a mutagen, carcinogen, and teratogen. Handle only in a biosafety fume hood while wearing a mask, chemically resistant gloves, protective clothing, and eye protection. Dispose of N,N-Mba and any contaminated consumables in a hazardous waste stream. Store at Temperature-20 °C .
Ethics statement
The protocols.io team notes that research involving animals and humans must be conducted according to internationally-accepted standards and should always have prior approval from an Institutional Ethics Committee or Board.
Before start
  • This protocol enables the visualization of individual mRNA transcripts in slices of fixed brain tissue. We anchor our brain slices via the [Gel and RNA Anchor Perfusion] protocol, but anchoring methods such as EASI-FISH or uniExM can also be used. See references for more information.

  • All enzymes are kept on ice.
Protocol Overview
5d
This protocol describes Multiplexed Fluorescence In Situ Hybridization (mFISH) on brain slices for retrospective transcriptomic analyses of neurophysiology and neuromorphology, with single transcript resolution. It is adapted from the Expansion Assisted Iterative FISH (EASI-FISH) protocol , where tissue is integrated into an expandable gel and transcript detection is amplified using the Hybridization Chain Reaction (HCR).
Citation
Wang Y, Eddison M, Fleishman G, Weigert M, Xu S, Wang T, Rokicki K, Goina C, Henry FE, Lemire AL, Schmidt U, Yang H, Svoboda K, Myers EW, Saalfeld S, Korff W, Sternson SM, Tillberg PW (2021). EASI-FISH for thick tissue defines lateral hypothalamus spatio-molecular organization. Cell.
10.1016/j.cell.2021.11.024
LINK
The first round of HCR RNA mFISH can be completed in one work week and each subsequent round can be completed in as few as two days.

Day 1
Gelation (~Duration03:30:00 ) and Proteinase K (ProK) Digestion (DurationOvernight )

Day 2
Hybridization with DNA probes(~Duration02:30:00 + DurationOvernight )

Day 3
Hybridizing hairpin amplifiers (~Duration06:00:00 + DurationOvernight ) or (~Duration09:00:00 )

Day 4
Expansion and imaging (~Duration02:30:00 )

Day 5
Probe removal and hybridization of next round's probes


Note
  • All aspects of this protocol should be performed RNase free. Whenever possible, handle samples and reagents in a PCR hood that has been sprayed thoroughly with RNase Away™. All tools and equipment should be sprayed thoroughly with RNase Away™ and all solutions should be kept RNase free. Where applicable, nuclease free water or Milli-Q water should be used. Wear a lab coat, mask, and gloves to prevent contamination. Spray gloves thoroughly with RNase Away™ and dry with a Kimwipe® regularly.

Note
The total volumes of the solutions are not specific. What is important is that the sample is completely submerged for these incubations.

5d
Preparation of gelled tissue slice
1d 7h
First, the brain tissue slice is embedded in a robust expandable hydrogel to anchor biomolecules and allow easy handling. Following gel polymerization, protein digestion via a ProK Solution allows isotropic expansion to occur.
Citation
Damstra H, Mohar B, Eddison M, Akhmanova A, Kapitein LK, Tillberg PW (2022). Visualizing cellular and tissue ultrastructure using Ten-fold Robust Expansion Microscopy (TREx). eLife.
10.7554/eLife.73775
LINK



Prepare solutions:
TREx monomer solution (9.4 mL):
ReagentsAmount
Sodium Acrylate2.7 mL
50% Acrylamide2.5 mL
2% N-N Methylenebisacrylamide24.6 µL
10x PBS1 mL
Nuclease Free Water3.4 mL
10% VA-044*110 µL
Total9.4 mL
* Note: Add VA-044 immediately before use; once it is added, it is referred to as "activated TREx".
The TREx solution recipe can also be found in the Materials section.

Safety information
TREx gels contain acrylamide, sodium acrylate, and N,N-methylenebisacrylamide. See Guidelines & Warnings for proper chemical safety. While handling TREx solutions, wear a mask, gloves, and protective clothing and dispose of TREx solutions in a dedicated hazardous waste stream.

50 mM ProK Buffer (100 mL):
ReagentsAmount
10% Triton X-1005 mL
1M Tris Base pH 85 mL
5M Sodium Chloride1 mL
0.5M EDTA200 µL
10% SDS3 mL
Water85.8 mL
Total100 mL
Note: Store 50 mM ProK Buffer at room temperature.
The ProK Buffer Solution recipe can also be found in the Materials section.
Incubate anchored brain slices in TREx monomer solution:
  • Place the anchored brain slices in a sterile container.
  • Incubate 3X Duration00:20:00 in activated TREx gel solution at TemperatureRoom temperature .
1h
Prepare the gelling mold:
  • Place a drop of activated TREx monomer solution on each end of a glass slide. Place a square coverslip (~22 mm x 22 mm) on each drop, leaving ~1 cm of space between the two coverslips. Place a drop of activated TREx monomer solution between the coverslips.
  • Coverslips can be layered to accommodate tissue slices of different thicknesses (see https://microscopy.duke.edu/guides/coverslip-thickness for average glass thickness). We use #1.5 coverslips for 150 μm tissue slices.
Prepare the tissue for gel formation:
  • Use a clean paintbrush to transfer the brain slice to the prepared glass slide, placing it between the coverslips on the drop of TREx monomer solution. Ensure it lays flat and no bubbles are trapped beneath the tissue slice.
  • Place another coverslip on top of the brain slice. If bubbles are present, use a clean razor blade to slightly lift the edge of the top coverslip and allow bubbles to escape.

An assembled gel mold with a coronal mouse brain slice in TREx gel monomer solution. No bubbles are present.

Polymerize the TREx gel in a humid chamber under inert atmosphere:
  • Create a humidified chamber by double bagging two Ziplock bags and then spraying the inside with RNase Away™ or nuclease-free water.
  • Place the gel molds on a large petri dish and tuck a Kimwipe saturated with RNase Away™ on the edge.
  • Place the petri dish and gel molds in the double bagged Ziplock bag and insert a tube connected to a nitrogen or argon line.
  • Purge both bags by pressing on each Ziplock bag at least three times and allow it to refill with nitrogen to ensure no oxygen is present.
  • Place this chamber in an oven and bake the prepared slice at Temperature45 °C for Duration01:30:00 or until the gels have solidified.
Note
  • The presence of bubbles can be an indication that the gel has solidified but isn't a consistent metric. We recommend lightly pressing on the surface of the top coverslip. If the gel solution beads or oozes out the sides of the mold, the gels should be returned to a humid chamber under inert atmosphere and continue to bake at Temperature45 °C .

1h 30m
Digest the gelled tissue with ProK Solution:
  • Fill a Microcentrifuge Tube with Amount742.5 µL Amount742.5 µL 50 mM ProK Buffer.
  • Using a clean razor blade, disassemble the polymerization chamber and cut the gel to the edge of the tissue.
  • Use a wetted clean paintbrush to gently dampen and lift the gelled tissue slice from the glass slide. Place the gelled tissue slice into the tube with ProK Buffer.
  • Add Amount7.5 µL Amount7.5 µL ProK (800 U/mL) to the tube.
  • Incubate the gelled tissue slice in ProK solution at Temperature37 °C for DurationOvernight .
  • Place the gelled tissue slice into fresh ProK solution at Temperature37 °C DurationOvernight .

The ProK Solution recipe can be found in the Materials section.


1d 3h
Wash out ProK Solution:
  • 3X Duration00:30:00 in 1X PBS at TemperatureRoom temperature .
  • Optional Pause Point: Store samples in 1X PBS at Temperature4 °C for up to 6 months.

1h 30m
FISH probe hybridization
1d 1h
Following gelation and digestion, DNA FISH probes are hybridized to RNA transcripts for genes of interest.

Equilibrate gelled tissue in HCR Probe Hybridization Buffer:
  • Thaw HCR Probe Hybridization Buffer to Temperature37 °C
  • Incubate samples for Duration01:00:00 in the HCR Probe Hybridization Buffer at Temperature37 °C .

Safety information
  • HCR Probe Hybridization Buffer contains 10-30% formamide. Formamide is toxic, carcinogenic, and teratogenic. Handle HCR Probe Wash Buffer in a well-ventilated area. Wear chemically resistant gloves, a mask, and protective clothing when handling.

  • Dispose of HCR Probe Hybridization Buffer and any contaminated consumables appropriately as hazardous waste.

1h
FISH probe hybridization
1d 1h
Prepare FISH probes for hybridization:
  • During HCR Probe Hybridization Buffer equilibration, thaw the appropriate FISH probes to TemperatureRoom temperature and prepare the FISH Probe Solution.

FISH Probe Solution Amount300 µL :
  • 10 nM of each FISH probe
  • 50 nM Rn28s
  • Remaining volume HCR Hybridization Buffer

Note
  • When designing HCR RNA mFISH, it is important to select orthogonal initiator sequences for each probe within the same round of mFISH. For example: If a probe with a B1 initiator sequence is used, no other B1 probes should be included in that round of mFISH.
  • We use a FISH probe against the Rn28s transcript for cell segmentation, but this is optional.
The FISH Probe Solution recipe can also be found in the Materials section.

FISH probe hybridization
1d 1h
Replace HCR Hybridization Buffer with FISH Probe Solution:
  • Incubate the sample in FISH Probe Solution DurationOvernight at Temperature37 °C
Note: Ensure the FISH Probe Solution volume completely covers the sample. Thicker samples will need a larger volume.
1d
HCR amplification
1d 6h 15m 30s
Next, HCR amplification is performed to amplify detected transcripts. Before hybridizing fluorescent amplifiers to the FISH probes, the sample must first be washed thoroughly to remove any remaining FISH probe.
Wash FISH probes from gelled tissue:
  • Thaw HCR Probe Wash Buffer at Temperature37 °C .
  • Wash samples 6X Duration00:20:00 in HCR Probe Wash Buffer at Temperature37 °C .
  • Wash samples 6X Duration00:30:00 in 1X PBS at Temperature37 °C .
  • Optional Pause Point: Store samples overnight in 1X PBS at Temperature4 °C for up to 6 months.
Safety information
  • HCR Probe Wash Buffer contains 10-30% formamide. Formamide is toxic, carcinogenic, and teratogenic. Handle probe wash buffer in a well-ventilated area. Wear gloves, a mask, and protective clothing when handling.

  • Dispose of HCR Probe Wash Buffer and any contaminated consumables appropriately as hazardous waste.

5h
Equilibrate gelled tissue in HCR Amplification Buffer:
  • Thaw HCR Amplification Buffer to TemperatureRoom temperature .
  • Remove 1x PBS and incubate samples in HCR Amplification Buffer for Duration01:00:00 at TemperatureRoom temperature .
1h
Snapcool HCR hairpins for signal amplification during HCR Amplification Buffer wash:
  • Our team uses a thermocycler (SimpliAmp™ Thermal Cycler) with the following program:
Phase 1:
Temperature95 °C Forever (skip this section)
Temperature95 °C Duration00:01:30
Phase 2 (14 cycles):
Temperature90 °C Duration00:01:00
Temperature85 °C Duration00:01:00
Temperature80 °C Duration00:01:00
Temperature75 °C Duration00:01:00
Temperature70 °C Duration00:01:00
Temperature65 °C Duration00:01:00
Temperature60 °C Duration00:01:00
Temperature55 °C Duration00:01:00
Temperature50 °C Duration00:01:00
Temperature45 °C Duration00:01:00
Temperature40 °C Duration00:01:00
Temperature35 °C Duration00:01:00
Temperature30 °C Duration00:01:00
Temperature25 °C Duration00:01:00
Phase 3:
Temperature20 °C Forever/hold
15m 30s
Prepare the Hairpin Solution:
  • Prepare the Hairpin Solution immediately before applying it to the samples. The hairpin initiator sequences must match the initiator sequences used in the FISH Probe Solution.
  • Wait to remove the hairpins from the SimpliAmp™ Thermal Cycler until immediately before adding them to the Hairpin Solution.

Hairpin solution Amount200 µL :
  • 30 nM H1 Hairpin
  • 30 nM H2 Hairpin
  • Remaining volume HCR Amplification Buffer
Incubate gelled tissue in the Hairpin Solution:
  • Incubate samples in the hairpin solution for Duration03:00:00 or DurationOvernight at TemperatureRoom temperature .
3h
Tissue expansion
3h
Following HCR amplification, samples are washed to remove excess hairpin, then expanded to enable high resolution imaging of individual transcripts.
Wash off hairpins:
  • 2X Duration00:30:00 in 5X SSCT at TemperatureRoom temperature .
  • 2X Duration00:30:00 in 0.5X SSCT at TemperatureRoom temperature .
2h
Expand gelled tissue:
  • 2X Duration00:30:00 in 1X PBS at TemperatureRoom temperature .
  • At this point, the gel will be approximately 3x-expanded. For lower expansion factors, use PBS concentrations greater than 1X.
1h
Imaging
At this point the sample is ready to be imaged. Our team uses an Olympus FV3000 confocal microscope or a Zeiss Lightsheet Z.1, but other fluorescence microscopy imaging systems can be used.
Mount the sample. The mounting protocol will vary depending on your imaging system.
Image the sample.
Probe Stripping with DNase
1d 2h
For carrying out additional rounds of mFISH, FISH probes and HCR amplifiers are removed using a DNase Solution and the process repeated for a new set of gene targets.
  • Optional Pause Point: Store samples in 1X PBS at Temperature4 °C up to 6 months.
Prepare DNase solutions for probe removal:
DNase Buffer Solution (750 μL):
AB
10x DNase Buffer75 μL
Water675 μL
Total Volume750 μL
The DNase Buffer Solution recipe can also be found in the Materials section.

DNase Solution (500 μL):
AB
DNase I20 μL
10X DNase Buffer 50 μL
Water430 μL
Total Volume500 μL
Note: We recommend making DNase Solution directly in each tube containing a gelled sample instead of making a stock. First add the water and 10X DNase Buffer, then spike the DNase I into each tube. Rock gently to combine. Keep DNase I TemperatureOn ice until addition to DNase Buffer.

Equilibrate gelled tissue in DNase Buffer:
  • Incubate the samples for Duration01:00:00 in DNase Buffer Solution at Temperature37 °C .
1h
Incubate gelled tissue in DNase I:
  • Incubate the samples for Duration01:00:00 in DNase Solution at Temperature37 °C .
  • Refresh the DNase Solution and keep at Temperature37 °C DurationOvernight .
1d 1h
After this step, the probes and hairpins have been removed. Subsequent rounds of mFISH can be performed with different probes. To begin a subsequent round, Go to .

Protocol references
Damstra H, Mohar B, Eddison M, Akhmanova A, Kapitein LK, Tillberg PW
Visualizing cellular and tissue ultrastructure using Ten-fold Robust Expansion Microscopy (TREx). eLife 2022, 11:e73775 doi: 10.7554/eLife.73775

Wang Y, Eddison M, Fleishman G, Weigert M, Xu S, Wang T, Rokicki K, Goina C, Henry FE, Lemire AL, Schmidt U, Yang H, Svoboda K, Myers EW, Saalfeld S, Korff W, Sternson SM, Tillberg PW.
EASI-FISH for thick tissue defines lateral hypothalamus spatio-molecular organization. Cell. 2021 doi: 10.1016/j.cell.2021.11.024

Citations
Step 1
Wang Y, Eddison M, Fleishman G, Weigert M, Xu S, Wang T, Rokicki K, Goina C, Henry FE, Lemire AL, Schmidt U, Yang H, Svoboda K, Myers EW, Saalfeld S, Korff W, Sternson SM, Tillberg PW. EASI-FISH for thick tissue defines lateral hypothalamus spatio-molecular organization
10.1016/j.cell.2021.11.024
Step 2
Damstra H, Mohar B, Eddison M, Akhmanova A, Kapitein LK, Tillberg PW. Visualizing cellular and tissue ultrastructure using Ten-fold Robust Expansion Microscopy (TREx)
10.7554/eLife.73775