Multiplexed Iterative FISH Experimental Protocol SOP002.v3.12

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 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.

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.

If using 4% , follow step 4. For 4% (v/v) , 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. 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 0h 5m 0s. Remove PBS and repeat this for a second wash.

ii. Pretreat tissue by washing in 4% SDS Clearing Solution (SDS-CS), once for 0h 5m 0s.

iii. To permeabilize the tissue, immerse the slip mounted tissue in 70% (vol/vol) ethanol 0h 5m 0s at 4°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 Room temperature PBS buffer for 0h 30m 0s to rehydrate the sample.

v. To bring the sample in sufficient contact with the coverslip surface, aspirate the PBS buffer and place in 37°C-45°C oven for 0h 5m 0s - 0h 10m 0s 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 Room temperature for 0h 10m 0s.

vii. Remove PFA from the sample and rinse with 1x PBS for 0h 5m 0s at RT, two times.

Using 4% PFA-fixed cells grown on coverslip (optionally, use 8-chamber well or similar) 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 0h 5m 0s at 4°C (recommended) in a Pyrex 60mm petri dish (Fisher 08-747A). (For faster results, sample can be incubated in EtOH for 1h 0m 0s at Room temperature).

ii. Alternatively, pipette 100µL permeabilization buffer (PBS-t) to each well and incubate at °C for 0h 10m 0s with gentle rocking.

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

iv. Aspirate rinse from the sample and let dry.

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.

Wash & equilibrate sample by immersing slip-mounted sample in 37°C pre-heated ~200µL Wash Buffer A for 0h 30m 0s.

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 125µL containing 1micromolar (µM) **

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 37°C in a humidified chamber for 18h 0m 0s -24h 0m 0s up to 36h 0m 0s .

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

Immerse slip in pre-heated 37°C Wash Buffer A for 30 min , two times.

Wash two times in 37°C pre-heated Encoding Wash Buffer (SSC-tw) for 5 min each.

Wash two times in Room temperature 1x PBS.

Wash sample for 0h 2m 0s** with de-gassed PA Solution. **adjust time based on sample size. For 100µm tissue slices, increase this to 3 hours.

Wash sample for 0h 2m 0s with PA Gel Solution and then remove.

Cast a thin PA film by adding 50µL - 100µL 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 1h 30m 0s at Room temperature .

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 37°C .

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

Incubate sample in 3mL with 10% at 20.000U/mL for 3h 0m 0s at 37°C.

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

Wash the sample on the coverslip twice with 1mL for 0h 5m 0s each wash at 37°C

Incubate with 3mL with 1% in a humidified chamber for 1h 0m 0s - 12h 0m 0s at 37°C, depending on the sample.

Wash the sample by immersing it in Wash Buffer B four times for 0h 5m 0s at Room temperature

Wash and equilibrate sample by immersing slip-mounted sample in 41°C pre-heated 200-500µL for 0h 30m 0s.

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 125µLcontaining 5micromolar (µM) -200micromolar (µM) (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 41°C in a humidified chamber for 18h 0m 0s -24h 0m 0s up to 36h 0m 0s .

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

Wash the sample in pre-heated 41°C Wash Buffer A for 0h 30m 0s, two times.

Wash two times in 41°C pre-heated Encoding Wash Buffer (SSC-tw) for 0h 5m 0s.

Wash two times in Room temperature 1x PBS.

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

Incubate sample in Wash Buffer C at 37°C for 0h 30m 0s.

Aspirate to remove Wash Buffer C.

Hybridize primary amplifier. Incubate the sample in a 125µL of 5nanomolar (nM) ***** in amplifier hybridization buffer for 0h 15m 0s ****** 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.

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

Hybridize secondary amplifier. Incubate the sample in a 125µL 5nanomolar (nM) ***** in amplifier hybridization buffer for 0h 15m 0s ****** 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.

Wash twice in Room temperature Wash C for 5 min each followed by a 0h 15m 0s - 0h 30m 0s wash in 37°C Wash C.

Perform MULTIPLEXED ITERATIVE FISH Imaging (Part 2) immediately or store sample for up to 24 hours in storage buffer at 4°C.

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.

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

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.

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 2mL over 0h 3m 0s to prime buffer to the sample.

b. Run additional 2.5mL over the sample for 0h 4m 0s.

c. Pause flow for 0h 15m 0s - 2h 0m 0s depending on sample size

        (10 µm = 15 min, 30 µm = 60 min, 100 µm = 120 min).

Readout Wash Buffer (Wash D)

a. Run 2mL over 0h 3m 30s to flush.

b. Run 2mL over 0h 10m 0s to wash.

Imaging Buffer

a. Run 2mL over 0h 10m 0s to run remaining wash D over sample and prime imaging buffer to the sample.

1. 2x SSC Wash Buffer (Wash B)

   a. Run 1mL over 0h 3m 0s to move 1mL imaging buffer over the sample.

Imaging. Pause fluidics and proceed with imaging.

TCEP Cleavage Buffer -

a. Run 2mL in 0h 3m 30s min to prime cleavage buffer to the sample.

b. Run 1mL over sample for 0h 5m 0s

c.Pause flow for 0h 10m 0s

2x SSC Wash Buffer (Wash B) -

a. Run 2mL in 0h 10m 0s to run cleavage buffer over sample and prime SSC buffer.

b. Run 2mL in 0h 4m 0s to rinse off cleavage buffer.

Repeat steps 54-60 for each readout round.

When all readout rounds are complete proceed with step 63.

DAPI Stain

a. Run 2mL DAPI in 2xSSC (Wash B) for 1h 0m 0s.

i.    Use `50µg/mL` DAPI for thick (40 μm) samples.



ii.    Use `1µg/mL` -`10µg/mL` DAPI for 10 μm samples.

2xSSC (Wash B) - 2mL for 0h 3m 30s to flush.

Imaging Buffer - 2mL in 0h 6m 0s then halt flow.

Proceed with Imaging.

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.

Readout Probe Hybridization.

a. Pipette 200µL in Readout Hybridization Buffer to sample and incubate at Room temperature for 0h 10m 0s.

b. Aspirate Readout Hybridization Buffer from the sample.

Wash away unbound probe by adding 200µL to sample for 5 min, two times. Additional washes may improve the result.

Dapi Stain. Add 200µL with DAPI nuclear stain (at 1μg/mL) to sample and incubate for 0h 30m 0s at 37°C .

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

Add 100µL -200µL to sample and mount to glass plate with clear nail polish.

Proceed with imaging.

Readout Probe Hybridization.

a. Pipette 200µL in Readout Hybridization Buffer to sample and incubate at Room temperature for 0h 10m 0s.

b. Aspirate Readout Hybridization Buffer from the chambers.

Wash away unbound probe by adding 200µL to sample for 5 min, two times. Additional washes may improve the result.

Add 100µL -200µL to sample.

Proceed with imaging of the round.

TCEP Cleavage Buffer – 100µL for 0h 15m 0s.

2x SSC Wash Buffer (Wash B) – 250µL each well, three times.

Repeat steps 81-85 for each probe set round.

Move on to step 88 when all rounds are complete.

Add 200µL with DAPI nuclear stain (at 1μg/mL) to sample and incubate for 0h 30m 0s at 37°C .

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

Proceed to Imaging of the Sample.