Multiplexed Iterative FISH Experimental Protocol SOP002.v3.12

Rory Kruithoff; Lei Zhou; Douglas Shepherd

Mar 03, 2023

Version 3

Multiplexed Iterative FISH Experimental Protocol SOP002.v3.12 V.3

DOI

dx.doi.org/10.17504/protocols.io.yxmvmk6k6g3p/v3

¹Arizona State University

Rory Kruithoff

Arizona State University

DOI: dx.doi.org/10.17504/protocols.io.yxmvmk6k6g3p/v3

Protocol Citation: Rory Kruithoff, Lei Zhou, Douglas Shepherd 2023. Multiplexed Iterative FISH Experimental Protocol SOP002.v3.12. protocols.io https://dx.doi.org/10.17504/protocols.io.yxmvmk6k6g3p/v3Version created by Rory Kruithoff

Manuscript citation:

- 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

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: February 27, 2023

Last Modified: March 03, 2023

Protocol Integer ID: 77717

Keywords: In-situ hybridization, FISH, fluorescence, RNA, iterative FISH, formamide, amplified probes, bDNA, branched DNA, fluidics, acrydite, linker probe, anchor probe, encoding probes, tissue, cells, thick tissue, clearing, digestion, polyacrylamide gel,

Abstract

This document, SOP002 - Multiplexed Iterative FISH Experimental Protocol, describes the process for in-situ fluorescence labeling of RNA transcripts in cells and tissues using a layered probe design, which allows for identity barcoding (MERFISH or similar).  This protocol also provides the option for signal amplification using Branched DNA [bDNA] amplification.  Iterative rounds of labeling and imaging are possible through the use of a readout probe with a cleavable disulfide (S-S) reporter molecule, a method that allows for minimal disruption to sample integrity between rounds. This document also describes cell and tissue preparation for RNA FISH as well as a system of mRNA anchoring using a specialized poly-t (locked nucleic acid, LNA) probe with an acrydite linker to bind mRNAs to a polyacrylamide (gel) matrix.  Clearing and digestion techniques are used to reduce cellular autofluorescence and increase the signal to noise ratio of the final data.  This protocol is strongly derived from Moffitt 2016 (https://doi.org/10.1016/bs.mie.2016.03.020) with some modifications with credit for a majority of this protocol due to Moffitt et al 2016.

Attachments

SOP002.v3.12 Multipl...

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Guidelines

v3.12 revision notes
i.Minor revisions to language and formatting to make compatible with current protocol changes per current fluidics programming (flow speeds, solution priming).Also adjusted some formatting to improve flow and readability. 
ii.Updated tissue re-hydration time.

Materials

Solution Preparation:

Wash Buffer A (40% Formamide Wash Buffer)

2xReagentSSC (20X), RNase-freeThermo FisherCatalog #AM9763  
1% (vol/vol) ReagentTween 20Sigma AldrichCatalog #P9416-100ML  
40% (vol/vol) ReagentFormamideThermo Fisher ScientificCatalog #AM9342  
Nuclease-free water 

Wash Buffer A Master Mix, 45 ml:

36.75 ml nuclease-free water
7.5 ml ReagentSSC (20X), RNase-freeThermo FisherCatalog #AM9763  
750 µl ReagentTween 20Sigma AldrichCatalog #P9416-100ML  
Add 40% ReagentFormamideThermo Fisher ScientificCatalog #AM9342   to prepare on demand

Wash Buffer B

2x ReagentSSC (20X), RNase-freeThermo FisherCatalog #AM9763  prepared in nuclease-free water

Wash Buffer C (10% Formamide Wash Buffer)

Nuclease-free water
2x ReagentSSC (20X), RNase-freeThermo FisherCatalog #AM9763  
10% (vol/vol) ReagentFormamideThermo Fisher ScientificCatalog #AM9342  

Wash Buffer C Master Mix, 49.5 ml:

44 ml nuclease-free water
5.5 ml ReagentSSC (20X), RNase-freeThermo FisherCatalog #AM9763  
Add 10% ReagentFormamideThermo Fisher ScientificCatalog #AM9342  formamide to prepare on demand

Saber Encoding Hybridization Buffer

Nuclease-free water
2x ReagentSSC (20X), RNase-freeThermo FisherCatalog #AM9763  
40% (vol/vol) ReagentFormamideThermo Fisher ScientificCatalog #AM9342  Formamide
0.1% (wt/vol) ReagentYeast tRNAThermo FisherCatalog #15401011  
1% (vol/vol) ReagentRNase Inhibitor, Murine - 15,000 unitsNew England BiolabsCatalog #M0314L  
1% (vol/vol) ReagentTween 20Sigma AldrichCatalog #P9416-100ML  
10% (wt/vol) ReagentDextransulfateSigma AldrichCatalog #D8906-100g  
Add 5-200 µM encoding probes depending on the size of the pool
Prepare on demand

Saber Encoding Hybridization Buffer Master Mix, 4.8 ml (for 8.0 ml prep with formamide added)

Nuclease-free water
800 µl ReagentSSC (20X), RNase-freeThermo FisherCatalog #AM9763  
320 µl ReagentYeast tRNAThermo FisherCatalog #15401011 reconstituted to 25mg/ml
80 µl ReagentRNase Inhibitor, Murine - 15,000 unitsNew England BiolabsCatalog #M0314L  
80 µl ReagentTween 20Sigma AldrichCatalog #P9416-100ML  
0.8 g ReagentDextransulfateSigma AldrichCatalog #D8906-100g  
Aliquot mix and store at -20°C
To prepare on demand, add 40% (vol/vol) ReagentFormamideThermo Fisher ScientificCatalog #AM9342 to master mix at time of use
Add 5-200 µM encoding probes depending on the size of the pool

Encoding Buffer Rinse (SSC-tw)

Nuclease-free water
2xReagentSSC (20X), RNase-freeThermo FisherCatalog #AM9763  
0.1% (vol/vol)ReagentTween 20Sigma AldrichCatalog #P9416-100ML  
Store at Room Temperature

PA Solution

Nuclease-free water
4% (vol/vol)Reagent40% Acrylamide/Bis Solution 19:1Catalog #1610144  
60 mMReagentTris (1 M), pH 8.0, RNase-freeThermo FisherCatalog #AM9856  
0.3 MReagentNaCl (5 M) RNase-freeThermo Fisher ScientificCatalog #AM9759  
One of the following:
For four-color experiments: 1:500 dilution 0.1µm-diameter light yellow beads ReagentFluorescent Light Yellow ParticlesSpheroTechCatalog #FP-0245-2 
For two-color experiments:1:200,000 dilution of 0.1µm-diameter carboxylate-modified orange fluorescent                           beadsReagentFluoSpheres™ Carboxylate-Modified MicrospheresThermo Fisher ScientificCatalog #F-8800  
De-gas solution before use
Prepare on demand

PA Gel

PA Solution including polymerizing agents:
0.03% (wt/vol)ReagentAmmonium PersulfateSigmaCatalog #A3678  
0.15% (vol/vol) TEMED 
Prepare on demand. Polymerizing agents will act rapidly. Make gel in small quantities (1ml) and right before use
Prepare on demand

Storage Buffer (SSC-SB)

Wash Buffer B
0.1% (vol/vol)ReagentRNase Inhibitor, Murine - 15,000 unitsNew England BiolabsCatalog #M0314L  
Store in aliquots at -20°C

Amplifier Hybridization Buffer

Nuclease-free water
2xReagentSSC (20X), RNase-freeThermo FisherCatalog #AM9763  
10% (vol/vol)ReagentFormamideThermo Fisher ScientificCatalog #AM9342  
0.1% (wt/vol)ReagentYeast tRNAThermo FisherCatalog #15401011  
1% (vol/vol)ReagentRNase Inhibitor, Murine - 15,000 unitsNew England BiolabsCatalog #M0314L  
10% (wt/vol)ReagentDextransulfateSigma AldrichCatalog #D8906-100g  
Prepare on demand

Amplifier Hybridization Buffer Master Mix, 7.2 ml (for 8.0 ml prep with formamide added)

Nuclease-free water
800 µlReagentSSC (20X), RNase-freeThermo FisherCatalog #AM9763  
320 µlReagentYeast tRNAThermo FisherCatalog #15401011  reconstituted to 25mg/ml
80 µlReagentRNase Inhibitor, Murine - 15,000 unitsNew England BiolabsCatalog #M0314L  
0.8 gReagentDextransulfateSigma AldrichCatalog #D8906-100g  
Aliquot mix and store at -20°C
To prepare on demand, add 10% (vol/vol) ReagentFormamideThermo Fisher ScientificCatalog #AM9342 to master mix at time of use
Add 5 nM amplifiers

Readout Hybridization Buffer

2xReagentSSC (20X), RNase-freeThermo FisherCatalog #AM9763  
10% (vol/vol)ReagentEthylencarbonat 98%Sigma AldrichCatalog #E26258  
0.1% (vol/vol)ReagentRNase Inhibitor, Murine - 15,000 unitsNew England BiolabsCatalog #M0314L  
Nuclease-free water
3 nM readout probes
Prepare on demand 

Wash Buffer D (Readout Wash Buffer)

2xReagentSSC (20X), RNase-freeThermo FisherCatalog #AM9763  
10% (vol/vol)ReagentEthylencarbonat 98%Sigma AldrichCatalog #E26258  
Store at Room Temperature

Imaging Buffer

2xReagentSSC (20X), RNase-freeThermo FisherCatalog #AM9763  
50 mMReagentTris (1 M), pH 8.0, RNase-freeThermo FisherCatalog #AM9856  
10% (wt/vol)ReagentD-( )-GlucoseSigma AldrichCatalog #DX0145-1  
2 mMReagentTroloxSigma AldrichCatalog # 238813  
0.5 mg/mlReagentGlucose oxidase Sigma AldrichCatalog #G2133  
40 µg/ml ReagentCatalaseSigmaCatalog #C30    
0.1% (vol/vol)ReagentRNase Inhibitor, Murine - 15,000 unitsNew England BiolabsCatalog #M0314L  
Nuclease-free water
Prepare on demand
Store under layer of Mineral Oil when using fluidics systemReagentMineral oilSigma AldrichCatalog #330779  

Cleavage Buffer

2xReagentSSC (20X), RNase-freeThermo FisherCatalog #AM9763  
50 mMReagentTris(2-carboxyethyl)phosphine hydrochloride solutionSigma AldrichCatalog #646547-10X1ML  
Prepare on demand

DAPI Staining Solution

50 µg/mlReagent4,6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI)Thermo Fisher ScientificCatalog #D1306  in Wash Buffer B for thick (40 µm) tissue
1-10 µg/ml Reagent4,6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI)Thermo Fisher ScientificCatalog #D1306 in Wash Buffer B for thin (10 µm) tissue
Prepare on demand

Permeabilization Buffer (PBS-t)

Nuclease-free water
1xReagentPBS - Phosphate-Buffered Saline (10X) pH 7.4Invitrogen - Thermo FisherCatalog #AM9625  
0.5% (v/v)ReagentTriton™ X-100Sigma AldrichCatalog #T8787-100ML  
Store at Room Temperature

Permeabilization Buffer Wash (PBS-tw)

Nuclease-free water 
1x ReagentPBS - Phosphate-Buffered Saline (10X) pH 7.4Invitrogen - Thermo FisherCatalog #AM9625  
0.1% (v/v)ReagentTween 20Sigma AldrichCatalog #P9416-100ML  
Store at Room Temperature

SDS Clearing Solution (SDS-CS)

Nuclease Free Water
4% ReagentSDSSigmaCatalog #75746  
200 mM ReagentBoric AcidSigmaCatalog #B0394 
pH to 8.5
Store at Room Temperature

  

Safety warnings

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.

Before start

All reagents for this protocol should be prepared sterile and RNase-free.
All incubation periods should be done in the dark.
Find reagent/buffer preparation instructions at index "Materials".

Quick Overview:

Part 1 - Tissue or Cell-Based Experiment Preparation
Step 1 - Coverslip functionalization
Step 2 - Mount, SDS pretreat and permeabilize sample
Step 3 - Hybridize linker (optional)
Step 4 - Wash away residual linker
Step 5 - Gel embed sample (optional)
Step 6 - Clear & digest sample (optional)
Step 7 - Hybridize encoding probes
Step 8 - Wash away residual encoding probes
Step 9 - Hybridize amplifiers (optional)

Part 2a - Multiplexed Iterative FISH Imaging with Fluidics System
Step 1 - Prepare solutions for imaging
Step 2 - Assemble fluidics system
Step 3 - MULTIPLEXED ITERATIVE FISH imaging protocol

Part 2b - Alternate MULTIPLEXED ITERATIVE FISH Imaging without Fluidics System
Step 1a: MULTIPLEXED ITERATIVE FISH imaging protocol - using coverslip mounted sample.
Step 1b: MULTIPLEXED ITERATIVE FISH imaging protocol - multiple hybridizations using chamber slip.
Step 2: Dapi stain the sample.
Step 3: Proceed to imaging of the sample.

Document Summary

Document Summary: This document, SOP002 - Multiplexed Iterative FISH Experimental Protocol, describes the process for in-situ fluorescence labeling of RNA transcripts in cells and tissues using a layered probe design, which allows for identity barcoding (MERFISH or similar) and/or signal amplification (Branched DNA [bDNA] amplification), along with a cleavable disulfide (S-S) reporter molecule, attached to a readout oligo, to allow for iterative rounds of labeling and imaging of the same sample with minimal disruption to sample integrity between rounds.This document also describes cell and tissue handling for the labeling process, and the RNA labeling process which uses an mRNA binding using a specialized poly-t (locked nucleic acid, LNA) probe with an acrydite linker to bind mRNAs to a polyacrylamide matrix and clearing techniques used to reduce cellular autofluorescence and increase the signal to noise ratio of the final data.

Part 1 - Tissue or Cell-Based Experiment Preparation

Part 1 of this protocol describes the steps to setup a multiplexed iterative FISH experiment for tissue or cell-based samples. These steps are focused on the biochemical requirements 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.

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

1h 5m

If using Concentration4 % PFA-fixed tissue  , follow step 4.  For Concentration4 % (v/v) PFA-fixed cells  ,  skip below to step 5.

Using 4% PFA-fixed tissue.  Note: For some tissue, it is simpler to mount directly to the coverslip after slicing.  In this case, mount and post-fix first then follow the remaining steps in order.

i.   Slice tissue and place slices in 1xPBS for Duration00:05:00  . Remove PBS and repeat this for a second wash.

ii.  Pretreat tissue by washing in 4% SDS Clearing Solution (SDS-CS), once for Duration00:05:00  .

iii. To permeabilize the tissue, immerse the slip mounted tissue in 70% (vol/vol) ethanol DurationOvernight   at Temperature4 °C   (recommended) in a Pyrex 60mm petri dish (Fisher 08-747A) or similar. (For faster results, sample can be incubated in EtOH for 1 hour at RT).

iv. Move tissue slices to functionalized (PDL-coated) coverslip, aspirate off the 70% ethanol and incubate in TemperatureRoom temperature   PBS buffer for Duration00:30:00   to rehydrate the sample.

v. To bring the sample in sufficient contact with the coverslip surface, aspirate the PBS buffer and place in Temperature37 °C  -Temperature45 °C   oven for Duration00:05:00   - Duration00:10:00   to dry any excess PBS buffer.  Monitor the progress closely. You will want to ensure the sample lying flat on the coverslip surface without drying the sample.

vi. Post-fix tissue to the coverslip by incubating in 4% PFA at TemperatureRoom temperature   for Duration00:10:00 ..

vii. Remove PFA from the sample and rinse with 1x PBS for Duration00:05:00   at RT, two times.

1h 15m

Using 4% PFA-fixed cells grown on coverslip (optionally, use 8-chamber well or similar)

i. To permeabilize the cells, immerse the slip mounted sample in 70% (vol/vol) ethanol DurationOvernight   at Temperature4 °C   (recommended) in a Pyrex 60mm petri dish (Fisher 08-747A). (For faster results, sample can be incubated in EtOH for Duration01:00:00   at TemperatureRoom temperature  ).

ii. Alternatively, pipette 100µL permeabilization buffer (PBS-t) to each well and incubate at Temperature0 °C   for Duration00:10:00   with gentle rocking.

iii. Rinse with TemperatureRoom temperature   permeabilization buffer rinse (PBS-tw).

iv. Aspirate rinse from the sample and let dry.

1h 15m

Using a hydrophobic pen, draw a barrier around your sample and let dry before hybridizations. You may want to add a very small volume of PBS during this process to tissue samples to prevent sample desiccation.

Part 1 - Step 3: Hybridize Linker (optional; use when gel embedding and digesting sample)

1d 12h 30m

Wash & equilibrate sample by immersing slip-mounted sample in Temperature37 °C   pre-heated ~Amount200 µL   Wash Buffer A for Duration00:30:00  .

30m

Assemble humidified chamber (empty pipette box with lid or otherwise that can house the sample-mounted coverslip with a single, saturated and folded paper used to line the inner edge of the chamber to prevent evaporation of probe solution).

Remove slip from Wash Buffer A and carefully wipe away excess buffer surrounding sample.

Dispense Amount125 µL Encoding Hybridization Buffer  containing Concentration1 micromolar (µM) linker  **
to your sample, replace the petri dish lid, parafilm the dish and place the dish with the sample in the humidified chamber. **Adjust concentration of the linker according to sample size.

Incubate at Temperature37 °C   in a humidified chamber for Duration18:00:00   -Duration24:00:00    up to Duration36:00:00   .

1d 12h

Part 1 - Step 4: Wash Away Residual Linker

1h 10m

Remove the hybridization buffer and carefully remove excess buffer surrounding sample.

Immerse slip in pre-heated Temperature37 °C   Wash Buffer A for 30 min , two times.

Wash two times in Temperature37 °C    pre-heated Encoding Wash Buffer (SSC-tw) for 5 min each.

Wash two times in TemperatureRoom temperature    1x PBS.

Part 1 - Step 5: Gel Embed Sample (optional - gel embed when clearing and digesting)

1h 34m

Wash sample for Duration00:02:00  ** with de-gassed PA Solution.  **adjust time based on sample size.  For 100µm tissue slices, increase this to 3 hours.

Wash sample for Duration00:02:00    with PA Gel Solution and then remove.

Cast a thin PA film by adding Amount50 µL gel solution  - Amount100 µL gel solution  to the sample and invert a smaller (25 mm) gel-slick coated coverslip onto the gel solutions being careful to avoid air bubbles. Adjust the volume and make sure your gel film is thin. Aspirate any extra gel solution away.

Allow casting for Duration01:30:00   at TemperatureRoom temperature   .

1h 30m

After casting, carefully remove the smaller coverslip from your sample. If the coverslip is stuck, you can loosen the coverslip by immersing in SDS-CS at Temperature37 °C   .

Part 1 - Step 6: Digest & Clear Sample (optional) (buffer and enzyme may vary depending on sample type)

15h 55m

Note: For lung tissue start at step 22.  Skip ahead to step 24 for brain tissue.

Incubate sample in Amount3 mL PBS  with Concentration10 % collagenase/elastase  at Concentration20.000 U/mL   for Duration03:00:00   at Temperature37 °C  . 

Wash the sample with a quick rinse of RT 1x PBS followed by two 5 min washes of 1x PBS atTemperatureRoom temperature  . 

Wash the sample on the coverslip twice with Amount1 mL SDS Clearing Solution (SDS-CS)   for Duration00:05:00   each wash at Temperature37 °C   

Incubate with Amount3 mL SDS Clearing Solution   with Concentration1 % Proteinase K  in a humidified chamber for Duration01:00:00   - Duration12:00:00   at Temperature37 °C  , depending on the sample.

13h

Wash the sample by immersing it in Wash Buffer B four times for Duration00:05:00   at TemperatureRoom temperature   

Part 1 - Step 7: Hybridize Encoding Probes

1d 12h 30m

Wash and equilibrate sample by immersing slip-mounted sample in Temperature41 °C   pre-heated Amount200-500 µL Wash Buffer A  for Duration00:30:00  . 

30m

Assemble a humidified chamber (an empty pipette box with lid or otherwise that can house the sample-mounted coverslip with a single, saturated and folded paper used to line the inner edge of the chamber to prevent evaporation of probe solution).

Remove the slip from Wash Buffer A and carefully wipe away the excess buffer surrounding the sample.

Dispense Amount125 µL Encoding Hybridization Buffer containing Concentration5 micromolar (µM)   -Concentration200 micromolar (µM) encoding probes   (depending on the number of unique encoding probes in the probe set and sample size) to your sample, replace the petri dish lid, parafilm the dish and place the dish with the sample in the humidified chamber.

Incubate at Temperature41 °C   in a humidified chamber for Duration18:00:00   -Duration24:00:00    up to Duration36:00:00   .

1d 12h

Part 1 - Step 8: Wash Away Residual Encoding Probes

1h 10m

Remove the hybridization buffer and carefully remove the excess buffer surrounding the sample.

Wash the sample in pre-heated Temperature41 °C    Wash Buffer A for Duration00:30:00  , two times.

30m

Wash two times in Temperature41 °C    pre-heated Encoding Wash Buffer (SSC-tw) for Duration00:05:00  .

Wash two times in TemperatureRoom temperature    1x PBS.

Part 1 - Step 9: Hybridize amplifiers (optional)

1h 25m

To label the gel embedded and cleared samples with primary and secondary amplifiers.

Incubate sample in Wash Buffer C at Temperature37 °C   for Duration00:30:00  . 

30m

Aspirate to remove Wash Buffer C.

Hybridize primary amplifier.  Incubate the sample in a  Amount125 µL droplet  of Concentration5 nanomolar (nM) primary amplifier * in amplifier hybridization buffer for  Duration00:15:00  ** in humidity-controlled 37°C incubator. 

*Amplifier concentration may need to be increased based on the thickness of your sample.
**Adjust incubation time based on sample size. For a 100µm tissue section, an overnight incubation is preferable for the primary amplifiers while the secondary amplifiers can be incubated for 5-6 hours, on the following day.

15m

Wash 3 times with Wash C for 5-10 min each at TemperatureRoom temperature  . 

Hybridize secondary amplifier.  Incubate the sample in a  Amount125 µL droplet  Concentration5 nanomolar (nM) secondary amplifier * in amplifier hybridization buffer for  Duration00:15:00  ** in humidity-controlled 37°C incubator. 

*Amplifier concentration may need to be increased based on the thickness of your sample.
**Adjust incubation time based on sample size. For a 100µm tissue section, an overnight incubation is preferable for the primary amplifiers while the secondary amplifiers can be incubated for 5-6 hours, on the following day.

15m

Wash twice in TemperatureRoom temperature   Wash C for 5 min each followed by a Duration00:15:00    - Duration00:30:00   wash in Temperature37 °C    Wash C.

45m

Perform MULTIPLEXED ITERATIVE FISH Imaging (Part 2) immediately or store sample for up to 24 hours in storage buffer at Temperature4 °C  . 

Part 2a - MULTIPLEXED ITERATIVE FISH Imaging with Fluidics System

Note
**The following steps (Part 2a) are used for the Full MULTIPLEXED ITERATIVE FISH protocol. For experiments that don’t use the fluidics system, move to Part 2B below.

Imaging for MULTIPLEXED ITERATIVE FISH involves multiple rounds of fluid exchange to hybridize, image, cleave and rinse samples. Automated fluid exchange and imaging approach is recommended. For setups lacking an automated fluidics exchange system, proceed to Part 2b.

Part 2a - Step 1: Prepare Solutions for Imaging

Prepare the following solutions with the corresponding volumes:

i.     Readout Hybridization Buffer (RHB)
ii.    Readout Wash Buffer (Wash D)
iii.   Imaging Buffer (store under mineral oil) (IB)
iv.   TCEP Cleavage Buffer (CB)
v.    2x SSC Wash Buffer (Wash B)
vi.   DAPI Staining Solution

Part 2a - Step 2: Assemble Fluidics System

Make sure that all tubing is properly connected. MULTIPLEXED ITERATIVE FISH probes and preparation time are costly so leaks need to be avoided at all costs.

Ensure the system is fully assembled, plugged in and turned on.

Double-check correctness of the details for the pump protocol for the MULTIPLEXED ITERATIVE FISH
Fluidics for the current project.

Load the sample to the flow cell and connect.

Carefully load all solutions to the proper reservoirs.

Part 2a - Step 3: MULTIPLEXED ITERATIVE FISH Imaging Protocol

1h 5m

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:

Readout hybridization buffer (with readout probes)

a.    Run Amount2 mL   over Duration00:03:00   to prime buffer to the sample.
b.    Run additional Amount2.5 mL   over the sample for Duration00:04:00  .
c.    Pause flow for Duration00:15:00   - Duration02:00:00   depending on sample size 
            (10 µm = 15 min, 30 µm = 60 min, 100 µm = 120 min).

2h 22m

Readout Wash Buffer (Wash D)
   a. Run   Amount2 mL    over Duration00:03:30   to flush.
   b. Run  Amount2 mL    over Duration00:10:00   to wash.

13m 30s

Imaging Buffer
a.  Run  Amount2 mL    over Duration00:10:00   to run remaining wash D over sample and prime imaging buffer to the sample.

10m

1. 2x SSC Wash Buffer (Wash B)
    a. Run Amount1 mL    over Duration00:03:00   to move 1mL imaging buffer over the sample.  

Imaging. Pause fluidics and proceed with imaging.

TCEP Cleavage Buffer - 
a. Run Amount2 mL    in Duration00:03:30   min to prime cleavage buffer to the sample.
b. Run Amount1 mL    over sample for  Duration00:05:00  
c.Pause flow for Duration00:10:00   

18m 30s

2x SSC Wash Buffer (Wash B) -
a. Run  Amount2 mL    in Duration00:10:00   to run cleavage buffer over sample and prime SSC buffer.
b. Run  Amount2 mL    in Duration00:04:00   to rinse off cleavage buffer. 

14m

Repeat steps 54-60 for each readout round.

When all readout rounds are complete proceed with step 63.

DAPI Stain 

a. Run Amount2 mL    DAPI in 2xSSC (Wash B) for Duration01:00:00  .
    i.    Use Concentration50 µg/mL   DAPI for thick (40 μm) samples.
    ii.    Use Concentration1 µg/mL   -Concentration10 µg/mL   DAPI for 10 μm samples.

2xSSC (Wash B) - Amount2 mL   for Duration00:03:30   to flush.

3m 30s

Imaging Buffer - Amount2 mL    in Duration00:06:00    then halt flow.

Proceed with Imaging. 

Part 2b - Alternate MULTIPLEXED ITERATIVE FISH Imaging without Fluidics

Note
The following steps are used for manual, iterative FISH without a fluidics system. For trial that uses the fluidics system, move to Part 2a (above).

For some MULTIPLEXED ITERATIVE FISH experiments, it may be simpler to proceed without the
fluidics system for imaging. Once you have hybridized probes and amplifiers if desired, readout probes can be hybridized and imaged in a single round or in multiple rounds if necessary. If you are hybridizing more than one round of readouts, proceed to Steps 1b-3.

Part 2b - Step 1a: MULTIPLEXED ITERATIVE FISH Imaging Protocol - Single Hybridization using coverslip mounted sample.

Readout Probe Hybridization.

a.    Pipette Amount200 µL 3nM readout probes  in Readout Hybridization Buffer to sample and incubate at TemperatureRoom temperature    for Duration00:10:00  .
b.    Aspirate Readout Hybridization Buffer from the sample.

10m

Wash away unbound probe by adding Amount200 µL RT Readout Wash Buffer D   to sample for 5 min, two times. Additional washes may improve the result.

Dapi Stain. Add Amount200 µL Wash Buffer B    with DAPI nuclear stain (at 1μg/mL) to sample and incubate for Duration00:30:00    at Temperature37 °C   .

30m

Remove the Dapi stain and wash with Wash Buffer B for 5 min, two times.

Add Amount100 µL   -Amount200 µL Imaging buffer  to sample and mount to glass plate with clear nail polish.

Proceed with imaging.

Part 2b - Step 1b: MULTIPLEXED ITERATIVE FISH Imaging - Multiple Hybridizations Using Chamber-slip

35m

Readout Probe Hybridization.

a.    Pipette Amount200 µL 3nM readout probes  in Readout Hybridization Buffer to sample and incubate at TemperatureRoom temperature    for Duration00:10:00  .
b.    Aspirate Readout Hybridization Buffer from the chambers.

10m

Wash away unbound probe by adding Amount200 µL RT Readout Wash Buffer D   to sample for 5 min, two times. Additional washes may improve the result.

Add Amount100 µL   -Amount200 µL Imaging buffer  to sample. 

Proceed with imaging of the round.

TCEP Cleavage Buffer – Amount100 µL   for Duration00:15:00  .

15m

2x SSC Wash Buffer (Wash B) – Amount250 µL   each well, three times.

Repeat steps 81-85 for each probe set round.

Move on to step 88 when all rounds are complete.

Part 2b - Step 2: Dapi Stain the Sample

40m

Add Amount200 µL Wash Buffer B    with DAPI nuclear stain (at 1μg/mL) to sample and incubate for Duration00:30:00    at Temperature37 °C   .

30m

Wash sample in Wash Buffer B for 5 min two times.

Part 2b - Step 3:

Proceed to Imaging of the Sample.

Public workspaceMultiplexed Iterative FISH Experimental Protocol SOP002.v3.12 V.3

Multiplexed Iterative FISH Experimental Protocol SOP002.v3.12 V.3