Sep 24, 2025

Public workspaceIterative RNA FISH Experimental Protocol SOP002.v.4.6

DOI
dx.doi.org/10.17504/protocols.io.3byl465o8go5/v1
Iterative RNA FISH Experimental Protocol SOP002.v.4.6
  • Rory Kruithoff1,
  • Douglas Shepherd1
  • 1Arizona State University
  • Human Cell Atlas Method Development Community
Rory Kruithoff
Icon indicating open access to content
QR code linking to this content
DOI: https://dx.doi.org/10.17504/protocols.io.3byl465o8go5/v1
Protocol Citation: Rory Kruithoff, Douglas Shepherd 2025. Iterative RNA FISH Experimental Protocol SOP002.v.4.6. protocols.io https://dx.doi.org/10.17504/protocols.io.3byl465o8go5/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
Created: September 17, 2025
Last Modified: September 24, 2025
Protocol Integer ID: 227465
Keywords: In-situ hybridization, FISH, fluorescence, RNA, iterative FISH, formamide, oligo probes, fluidics, acrydite linker, anchor probe, encoding probes, tissue, cells, tissue, clearing, digestion, polyacrylamide, gel, tissue preparation for rna fish, rna fish, multiplexed iterative fish, experimental situ fluorescence labeling of mrna, locked nucleic acid , in-situ fluorescence labeling, dna, tissue preparation, readout probe, iterative rna fish experimental protocol, iterative rounds of rna fish labeling, rna fish labeling, situ fluorescence labeling of rna transcript, single molecule rna fish, situ fluorescence labeling, rna transcript, reporter molecule, tissue processing, situ labeling method, imaging of the same sample, imaging, smfish
Funders Acknowledgements:
Chan Zuckerberg Initiative
Grant ID: DAF2022-252554
National Institute of Mental Health
Grant ID: RF1MH128867
Abstract
This document, SOP002 - Iterative RNA FISH Experimental Protocol, describes the process for in-situ fluorescence labeling of RNA transcripts in cells and tissues using a layered (encoding + readout) probe design. This protocol can be used for single molecule RNA FISH or for high-throughput identity barcoding (MERFISH). This protocol uses a cleavable disulfide (S-S) reporter molecule, attached to a readout oligo, to allow for iterative rounds of RNA FISH labeling and imaging of the same sample with minimal disruption to sample integrity between rounds.  For sufficient singal:noise, samples are gel embedded, cleared and photobleached.  Tissue processing, gel embedding, photobleaching and clearing are described in addition to the in-situ labeling method. This protocol is strongly derived from Moffitt 2016 (https://doi.org/10.1016/bs.mie.2016.03.020) with some modifications. Credit for a majority of this protocol due to Moffitt et al 2016.

This protocol is an updated version of "Multiplexed Iterative FISH Experimental Protocol SOP002.v3.12 V.3 dx.doi.org/10.17504/protocols.io.yxmvmk6k6g3p/v3." This version focuses on efficient experimental flow, includes some chemistry changes, and incorporates a title change as this basic overall method can be used interchangeably with MERFISH and smFISH, either single color, or multiplexed.
Attachments
Icon for SOP002.v4.6 Iterative RNA FISH protocol.pdf
SOP002.v4.6 Iterativ...
582KB
Guidelines
**All reagents for this protocol should be prepared sterile and RNase-free.
**All incubation periods should be done in the dark.
**Reagent/buffer preparation instructions follow protocol below.
**Safety note:  This protocol uses formamide which is a teratogen and can cause developmental malformation.  Always work in a fume hood with formamide to avoid inhalation and avoid physical contact.
Materials
Wash Buffer A (40% Formamide Wash Buffer)
● 2x SSC Buffer (Fisher, AM9763)
● 1% (vol/vol) Tween 20 (Sigma, P9416-100ML)
● 40% (vol/vol) Formamide (Fisher, AM9342) 
● Nuclease-free water
Wash Buffer A Master Mix, 45mL (for 75mL final volume with FA):
● 36.75mL nuclease-free water
● 7.5mL 20x SSC Buffer (Fisher, AM9763)                    
● 750µL Tween 20 (Sigma, P9416-100ML)
● Add 40% formamide (FA) to prepare on demand (Fisher, AM9342) 
Wash Buffer B
● 2x SSC buffer (Fisher, AM9763) prepared in nuclease-free water.
Saber Encoding Hybridization Buffer
● Nuclease-free water
● 2x SSC Buffer (Fisher, AM9763)                    
● 40% (vol/vol) Formamide (Fisher, AM9342)
● 0.1% (wt/vol) Yeast tRNA (Life Technologies, 15401011)            
● 1% (vol/vol) Murine RNase Inhibitor (New England Labs, M0314L)
● 1% (vol/vol) Tween 20 (Sigma, P9416-100ML)
● 10% (wt/vol) Dextran sulfate (Sigma, D8906-100g)
● Add encoding probes at 5-200µM final concentration, depending on the size of the pool.
● Prepare on demand.
                 
Saber Encoding Hybridization Buffer Master Mix, 4.8mL (for 8.0mL prep with formamide added)
● Nuclease-free water
● 800µL 20x SSC Buffer (Fisher, AM9763)                    
● 320µL Yeast tRNA solution (Life Technologies, 15401011, reconstituted to 25mg/mL)            
● 80µL Murine RNase Inhibitor (New England Labs, M0314L)
● 80µL Tween 20 (Sigma, P9416-100ML)
● 0.8g Dextran sulfate (Sigma, D8906-100g)
● Aliquot mix and store at -20°C. 
● To prepare on demand, add 40% (vol/vol) Formamide (Fisher, AM9342) to master mix at time of use.
● Add encoding probes at 5-200µM final concentration, depending on the size of the pool.
Encoding Buffer Rinse (SSC-tw)
● Nuclease-free water
● 2x SSC (Fisher, AM9763)                    
● 0.1% (vol/vol) tween 20 (Sigma, P9416-100ML)
● Store at RT
PA Monomer Solution
● Nuclease-free water
● 4% (vol/vol) 19:1 acrylamide/bis-acrylamide (Bio-Rad Laboratories, 1610144)
● 60mM Tris-HCl pH8 (Fisher, AM9856)
● 0.3M NaCl (Fisher, AM9759)
● De-gas solution before use.
● Prepare on demand.
PA Gel
● PA monomer solution including polymerizing agents:
  • 0.03% (wt/vol) ammonium persulfate (Sigma A3678)
  • 0.15% (vol/vol) TEMED
  • **Add desired concentration of fluorescent beads for image registration
  • **Prepare on demand. 
Polymerizing agents will act rapidly.  Make gel in small quantities (1mL) and right before use.
o Optional:  Add fluorescent beads for image registration purposes, as needed.
● Prepare on demand.
Storage Buffer (SSC-SB)
● Wash Buffer B
● 0.1% (vol/vol) murine RNase Inhibitor (New England Labs, M0314L)
● Store in aliquots at -20°C.
5x SSC Storage Buffer (5xSSC-SB)
● 5x SSC buffer prepared in nuclease free water
● 3mg/mL Poly(vinylsulfonic acid, sodium salt)solution (Sigma, 278424)
● Prepare on demand
Readout Hybridization Buffer (RHB)
● 2x SSC buffer (Fisher, AM9763)
● 10% (vol/vol) ethylene carbonate (Sigma, E26258)
● 0.1% (vol/vol) Murine RNase Inhibitor (New England Labs, M0314L)
● Nuclease-free water
● Add readout probes at 3nM final concentration.
● Prepare on demand.
Readout Wash Buffer (RWB)
● 2x SSC Buffer (Fisher, AM9763)                                 
● 10% (vol/vol) ethylene carbonate (Sigma, E26258)
● 0.1% triton X-100
● Store at RT.
Trolox Quinone Imaging Buffer
  • 10% >500µM Trolox Quinone (see preparation instruction below)
  • 0.2% 2.5M PCA - 3,4-Dihydroxybenzoic acid (386mg/mL in methanol) (Sigma, 37580)
  • 0.5% Trolox (100mg/mL in methanol) (Sigma, 238813)
  • 0.5% 1M NaOH (Sigma, S5881)
  • 0.3% PVSA (Sigma, 278424)
  • Prepared in 2x SSC buffer (Invitrogen, AM9763)
Trolox Quinone Preparation Instruction
  • 100mM Trolox dissolved in methanol (250mg Trolox in 10mL methanol).
  • Add 0.4mL 100mM Trolox to 20mL 2x SSC buffer, 8x, and vortex to mix well.
  • Bake for 30min at 254nm in UVP-CX2000 or similar.
  • Remove aliquot and measure absorbance at 255nm. 
  • Repeat baking step until absorbance is 1.63 or greater for ~500µM Trolox Quinone.
  • Combine aliquots and measure the final combined absorbance.
  • Prepare 4mL aliquots and store at -20°C.
Cleavage Buffer
● 2x SSC Buffer (Fisher, AM9763)
● 50mM Tris(2-carboxyethyl) phosphine [TCEP] (Sigma, 646547)
● Prepare on demand.
DAPI Staining Solution
● 50µg/mL DAPI stain (Fisher, D1306) in Wash Buffer B for thick (40µm) tissue.
● 1-10µg/mL DAPI stain (Fisher, D1306) in Wash Buffer B for thin (10µm) tissue.
● Prepare on demand.
Permeabilization Buffer (PBS-t)
● Nuclease-free water
● 1x PBS Buffer (Invitrogen, AM9625)
● 0.5% (v/v) Triton X-100 (Sigma, T8787-100mL)
● Store at RT
Permeabilization Buffer Wash (PBS-tw)
● Nuclease-free water
● 1x PBS Buffer (Invitrogen, AM9625),
● 0.1% (v/v) Tween 20 (Sigma, P9416-100ML)
● Store at RT
SDS Clearing Solution (SDS-CS)
● 1x PBS
● 4% SDS (Sigma, 75746)
● Nuclease Free Water
● Store at RT
PFA Quenching solution (NH4CL)
  • 100mM Ammonium Chloride in nuclease-free water.
Troubleshooting
Safety warnings
**Safety note:  This protocol uses formamide which is a teratogen and can cause developmental malformation.  Always work in a fume hood with formamide to avoid inhalation and avoid physical contact.
Part 1 – Tissue or Cell-Based Experiment Preparation
Description: Part 1 of this protocol describes the steps to setup an iterative RNA FISH experiment for tissue or cell-based samples. These steps are focused on the biochemical requirement for tissue or cell preparation, probe hybridization and imaging.  This protocol does not cover the requirements of the microscope for imaging. Additional detail can for the imaging setup can be found at https://doi.org/10.1016/bs.mie.2016.03.020.

**All reagents for this protocol should be prepared sterile and RNase-free.
**All incubation periods should be done in the dark.
**Reagent/buffer preparation instructions follow protocol below.
**Safety note:  This protocol uses formamide which is a teratogen and can cause developmental malformation.  Always work in a fume hood with formamide to avoid inhalation and avoid physical contact.
Part 1, Step 1: Coverslip Functionalization Refer to current version of SOP003 for protocol on Coverslip Functionalization. PDL-coated coverslips are preferable as tissue can be post-fixed to the coating using 4% PFA.
Part 1, Step 2: Mount, SDS Pretreat and Permeabilize Sample
Part 1, Step 2a: Using 4% PFA-fixed and OCT-embedded tissue.  i. Slice tissue and blot tissue slice to a sample coverslip. Leave tissue slice on coverslip for 5 min in cryostat to re-freeze the sample to the coverslip surface. ii. Post-fix the sample to the coverslip using RT PFA for 10min, quench PFA with 100mM Ammonium chloride for 5-20 min then rinse with 1x PBS 3x for 5 min at RT. iii. Pretreat tissue:
1. (optional – tissue dependent) Immerse tissue in 5-6 drops of 3% peroxide solution at RT for 10 min then rinse sample with then rinse out well with 1x PBS 3x at RT.
2. Incubate in 4% SDS Clearing Solution (SDS-CS) for 5 minutes at RT then rinse out well with 1x PBS 3x for 5 min at RT. iv. To permeabilize the tissue, immerse the slip mounted tissue in 70% (vol/vol) ethanol overnight at 4°C (recommended) in a Pyrex 60mm petri dish. 
**For faster results, sample can be incubated in 70% EtOH for 1 hour at RT. v. Following permeabilization, aspirate off the 70% ethanol and move to step c. vi. Rehydrate sample with ethanol series of 50%, 30% and 10% EtoH in nuclease free water.
Part 1, Step 2b: Using 4% PFA-fixed cells grown on coverslip (optionally, use 8-chamber well or similar) i. Pretreat cells for 2min at RT in 2% SDS clearing solution (50% diluted 4% clearing solution in 1x PBS). ii. Rinse the sample 3x for 5 min in 1x PBS solution. iii. To permeabilize the cells, immerse the slip mounted sample in 70%(vol/vol) ethanol overnight at 4°C (recommended) in a Pyrex 60mm petri dish (Fisher 08-747A). (For faster results, sample can be incubated in EtOH for 1 hour at RT).  iv. Aspirate rinse from the sample and let dry.
Alternative permeabilization method for cells:
1. Pipette 100µL permeabilization buffer (PBS-t) and incubate at RT for 10 min with gentle rocking.
2. Rinse with permeabilization buffer rinse (PBS-tw) and rinse again with 1x PBS.
3. Let the sample dry and move on to step c.
Part 1, Step 2c:
Using a hydrophobic pen, draw a barrier around your sample and let the barrier dry before hybridizations.  You may want to add a very small volume of PBS to a tissue sample during this process to prevent tissue sample desiccation. 1) Using a hydrophobic pen, draw a barrier around your sample and let the barrier dry before hybridizations. You may want to add a very small volume of PBS to a tissue sample during this process to prevent tissue sample desiccation.
Part 1, Step 3: Hybridize anchor probe RNA linker (use linker when gel embedding).
  1. Wash & equilibrate sample by immersing slip-mounted sample in 52°C pre-heated 100µL-250µL Wash Buffer A for 5-30 min.
  2. Assemble a humidified chamber per figure 1.
  3. Remove Wash Buffer A from the sample.
  4. Dispense 125μL 47°C prewarmed Encoding Hybridization Buffer containing 1µM** linker to your sample, add an overlay coverslip and assemble the sample in the hybridization chamber per figure 1.
Part 1, Step 4: Wash Away Residual Linker
  1. Remove the hybridization buffer and carefully remove the excess buffer surrounding the sample. 
  2. Wash the sample in pre-heated 47°C Wash Buffer A for 30 min, two times.
  3. Wash two times in a 47°C pre-heated Encoding Wash Buffer (SSC-tw) for 5 mins, two times.
  4. Rinse two times with RT 1x PBS.
Part 1, Step 5: Gel Embed Sample (optional – gel embed when clearing and digesting)
  1. Incubate sample for 2 min with PA Gel Solution and remove.
  2. Cast a thin PA film by adding ~50-100µL gel solution (including fluorescent beads) to the sample and then invert a smaller (25mm-diameter) Gel Slick (Lonza, 50640) coated coverslip overlay onto the gel solution, being careful to avoid air bubbles.  Adjust the gel solution volume and make sure your gel film is thin by aspirating any extra gel solution away.  You can also carefully overlay a Kim wipe to blot extra gel solution from the sample.
  3. Allow casting for 1.5 hour at RT.
  4. After casting, carefully remove the overlay coverslip from your sample.  If the coverslip is stuck, you can loosen the coverslip by immersing SDS-CS at 37°C.
Part 1, Step 6: Photobleach and Clear Sample
  1. Depending on the tissue type (e.g. lung tissue), you may need to modify the clearing process.  For lung tissue, start with step Part 1, step 6.2.2.1 - 6.2.2.3. For brain tissues, skip to Part 1, step 6.3.
2. (optional for lung tissue) Enzymatic digestion
2.1. Incubate sample in 3mL PBS with 10% collagenase/elastase at 20,000U/mL for 3 hours at 37°C.
2.2. Wash sample with a quick rinse of RT 1x PBS followed by two 5min washes of 1x PBS at RT.
3. (optional, recommended) Photo bleach the sample overnight in 5x SSC storage buffer with 3mg/mL pvsa and protected using a UV filter and aluminum foil.
4. Wash the sample on the coverslip twice with 1mL SDS Clearing Solution (SDS-CS) for 5 min each wash at 37°C. Incubate with 3-5mL prewarmed 37°C SDS Clearing Solution with 1% Proteinase K in a humidified chamber for 1-24h at 37°C, depending on the sample. 
5. Wash the sample by immersing it in Wash Buffer B 3 to 4 times for 5 min each.
6. Rinse sample in 2x SSC, 3x.
Part 1, Step 7:  Hybridize Encoding probes.
  1. Wash & equilibrate sample by immersing slip-mounted sample in 52°C pre-heated 100µL-250µL Wash Buffer A for 5-30 min.
  2. Assemble a humidified chamber per figure 1.
  3. Remove Wash Buffer A from the sample.
  4. Dispense 125μL 47°C prewarmed Encoding Hybridization Buffer containing 5-200**µM encoding probes (depending on the number of unique encoding probes in the probe set and sample size) and add an overlay coverslip and assemble the sample in the hybridization chamber per figure 1. **Adjust the concentration of encoding probe depending on the sample size and thickness.
  5. Incubate at 43°C** in a humidified chamber for 18-24 hours, up to 72 hours.
**Note: hybridization temperature may need to be adjusted based on Tm of probes as designed. PaintSHOP probes hybridized at 43°C; Moffitt/Zhuang probes hybridized at 37°C.
Part 1, Step 8: Wash Away Residual Encoding Probes.
  1. Remove the hybridization buffer and carefully remove the excess buffer surrounding the sample. 
  2. Wash the sample in pre-heated 47°C Wash Buffer A for 30 min, two times.
  3. Wash two times in a 47°C pre-heated Encoding Wash Buffer (SSC-tw) for 5 mins, two times.
  4. Rinse two times with RT 1x PBS.

Figure 1. Hybridization chamber rendering.  For hybridization incubations, it is critical to prevent evaporation. For hybridizations, pipet the hybridization buffer on the sample then overlay with a cleaned and RNase-pretreated 25mm coverslip (or parafilm).  Place the sample coverslip in a 60mm petri dish (Fisher 08-747A), replace the petri dish lid then Parafilm the edges of the dish to prevent evaporation.  Wet a rolled paper towel and place in a clean, empty pipet tip box along with a sample stage which keeps the sample away from any standing water.

Part 2 – ITERATIVE RNA FISH Imaging with Automated Fluidics System
Description:  Imaging for ITERATIVE RNA FISH involves multiple rounds of fluid exchange to hybridize, image, cleave and rinse samples.  Automated fluid exchange and imaging approach is recommended.
Part 2, Step 1: Prepare Solutions for fluidics.
i. Readout Hybridization Buffer ii. Readout Wash Buffer iii. Imaging Buffer (store under mineral oil) iv.TCEP Cleavage Buffer v. 2x SSC Wash Buffer (Wash B) vi.DAPI Staining Solution (optional)

Suggested buffer volumes for use with pump-driven fluidics setup:
● Imaging buffer - 2.5mL per round. 
● TCEP Cleavage buffer - 4.5mL per round.
● RWB - 4.5mL per round.
Part 2, Step 2: Assemble Fluidics System
  1. Pre-rinse the fluidics setup following SOP002a before use.
  2. Make sure that all tubing is properly connected.  MULTIPLEXED ITERATIVE FISH probes and sample preparation time are costly so leaks need to be avoided.
  3. Ensure the system is fully assembled, plugged in and turned on.
  4. Double-check correctness of the details for the pump program for the MULTIPLEXED ITERATIVE FISH Fluidics for the current project.
  5. Load the sample to the flow cell and connect.
  6. Carefully load all solutions to the proper reservoirs.
  7. Prime the solutions using the solution pre-treatment program to reduce bubbles in lines and bring solutions to the distribution manifold.
Part 2, Step 3: ITERATIVE RNA FISH Imaging Protocol. Once the fluidics system is setup, solutions are prepped and loaded and the sample is in place in the chamber, an automated program should run the following cycle.
1. SSC Buffer
1a. Prime SSC 0.5mL/min
1b. Flow 3mL SSC buffer at 0.5mL/min.
2. Readout hybridization buffer (including readout probes)
2a. Prime RHB (0.5mL/min)
2b.Flow 2mL over sample at 0.4mL/min
2c. Pause flow for 15-120 mins, depending on sample size (10 µm tissue = 30 min, 30 µm = 60 min, 100 µm = 120 min).
3. Readout Wash Buffer
3a. Prime RWB (0.5mL/min)
3b. Flow 2.5mL over sample at 0.2mL/min)
4. SSC Buffer
4a.Prime SSC Buffer (0.5mL/min)
4b. Flow 2.5mL over sample (0.2mL/min)
5. Imaging Buffer 
5a. Flow 2mL IB over sample (0.2mL/min)
5b. Flow and additional 1mL IB over the sample (0.5mL/min).
5c. Pause flow for 15 mins to equilibrate sample.
6. Imaging.  Pause fluidics and proceed with imaging.
7. TCEP Cleavage Buffer 
7a. Prime CB (0.5mL/min)
7b. Flow 1.3mL CB over sample (0.13mL/min)
7c. Pause flow for 10 min.
8. SSC Buffer
8a. Prime SSC (0.5mL/min)
8b. Flow 3mL SSC buffer at 0.5mL/min
9. Repeat steps Part 2, Step 3.1-3.8 for each readout round.

When all readout rounds are complete proceed with steps 10-13.

10. DAPI Stain (optional)
10a. Wash 2mL DAPI in 2xSSC (Wash B) for 60 min.
- Use 50µg/mL for thick (100µm) samples.
- Use 1-10µg/mL for 10µm samples. 11. SSC Buffer
11a. Prime SSC Buffer (0.5mL/min)
11b. Flow 2.5mL over sample (0.2mL/min)
12. Imaging Buffer 
12a. Flow 2mL IB over sample (0.2mL/min)
12b. Flow and additional 1mL IB over the sample (0.5mL/min).
12c. Pause flow for 15 mins to equilibrate the sample 13. Imaging.  Pause fluidics and proceed with imaging.


Protocol references
-Hershberg, E. A., Close, J. L., Camplisson,C. K., Attar, S., Chern, R., Liu, Y., ... & Beliveau, B. J. (2020). PaintSHOP enables the interactive design of transcriptome-and genome-scale oligonucleotide FISH experiments. bioRxiv.
-Moffitt, J. R., Hao, J., Bambah-Mukku, D., Lu, T., Dulac, C., & Zhuang, X. (2016). High-performance multiplexed fluorescence in situ hybridization in culture and tissue with matrix imprinting and clearing. Proceedings of the National Academy of Sciences, 113(50), 14456-14461. https://doi.org/10.1073/pnas.1617699113
-Moffitt, J. R., & Zhuang, X. (2016). RNA imaging with multiplexed error-robust fluorescence in situ hybridization (MERFISH). In Methods in enzymology (Vol. 572, pp. 1-49). Academic Press. https://doi.org/10.1016/bs.mie.2016.03.020.
-Stellaris RNA FISH protocol for frozen tissues: https://biosearchassets.blob.core.windows.net/assets/bti_stellaris_protocol_frozen_tissue.pdf