TITLE: Multiplexed Iterative FISH Experimental Protocol SOP002.v3.12
AUTHORS: Rory Kruithoff, Lei Zhou, Douglas Shepherd

[DESCRIPTION]
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.

[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.

[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.

[STEPS]
SECTION: Part 1 - Tissue or Cell-Based Experiment Preparation
2. 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.
SECTION: Part 1 - Step 1: Coverslip Functionalization
3. 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.
SECTION: Part 1 - Step 2: Mount, SDS Pretreat and Permeabilize Sample
4. If using 4 % , follow step 4.  For 4 % (v/v) ,  skip below to step 5.
SECTION: Part 1 - Step 2: Mount, SDS Pretreat and Permeabilize Sample
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 5 min. Remove PBS and repeat this for a second wash.

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

iii. To permeabilize the tissue, immerse the slip mounted tissue in 70% (vol/vol) ethanol 5 min 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 30 min 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 5 min - 10 min 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 10 min.

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


SECTION: Part 1 - Step 3: Hybridize Linker (optional; use when gel embedding and digesting sample)
8. Wash & equilibrate sample by immersing slip-mounted sample in 37 °C pre-heated ~200 µL Wash Buffer A for 30 min.

SECTION: Part 1 - Step 3: Hybridize Linker (optional; use when gel embedding and digesting sample)
9. 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).
SECTION: Part 1 - Step 3: Hybridize Linker (optional; use when gel embedding and digesting sample)
10. Remove slip from Wash Buffer A and carefully wipe away excess buffer surrounding sample.
SECTION: Part 1 - Step 3: Hybridize Linker (optional; use when gel embedding and digesting sample)
11. Dispense 125 µL containing 1 micromolar (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.
SECTION: Part 1 - Step 3: Hybridize Linker (optional; use when gel embedding and digesting sample)
12. Incubate at 37 °C in a humidified chamber for 1080 min -1440 min  up to 2160 min .

SECTION: Part 1 - Step 4: Wash Away Residual Linker
13. Remove the hybridization buffer and carefully remove excess buffer surrounding sample.
SECTION: Part 1 - Step 4: Wash Away Residual Linker
14. Immerse slip in pre-heated 37 °C Wash Buffer A for 30 min , two times.

SECTION: Part 1 - Step 4: Wash Away Residual Linker
15. Wash two times in 37 °C  pre-heated Encoding Wash Buffer (SSC-tw) for 5 min each.

SECTION: Part 1 - Step 4: Wash Away Residual Linker
16. Wash two times in Room temperature  1x PBS.

SECTION: Part 1 - Step 5: Gel Embed Sample (optional - gel embed when clearing and digesting)
17. Wash sample for 2 min** with de-gassed PA Solution.  **adjust time based on sample size.  For 100µm tissue slices, increase this to 3 hours.

SECTION: Part 1 - Step 5: Gel Embed Sample (optional - gel embed when clearing and digesting)
18. Wash sample for 2 min  with PA Gel Solution and then remove.

SECTION: Part 1 - Step 5: Gel Embed Sample (optional - gel embed when clearing and digesting)
19. Cast a thin PA film by adding 50 µL - 100 µL 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.

SECTION: Part 1 - Step 5: Gel Embed Sample (optional - gel embed when clearing and digesting)
20. Allow casting for 90 min at Room temperature .

SECTION: Part 1 - Step 5: Gel Embed Sample (optional - gel embed when clearing and digesting)
21. 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 .

SECTION: Part 1 - Step 6: Digest &amp; Clear Sample (optional) (buffer and enzyme may vary depending on sample type)
25. Wash the sample on the coverslip twice with 1 mL  for 5 min each wash at 37 °C 

SECTION: Part 1 - Step 6: Digest &amp; Clear Sample (optional) (buffer and enzyme may vary depending on sample type)
26. Incubate with 3 mL  with 1 % in a humidified chamber for 60 min - 720 min at 37 °C, depending on the sample.

SECTION: Part 1 - Step 6: Digest &amp; Clear Sample (optional) (buffer and enzyme may vary depending on sample type)
27. Wash the sample by immersing it in Wash Buffer B four times for 5 min at Room temperature 

SECTION: Part 1 - Step 6: Digest &amp; Clear Sample (optional) (buffer and enzyme may vary depending on sample type)
24. Wash the sample with a quick rinse of RT 1x PBS followed by two 5 min washes of 1x PBS atRoom temperature. 

SECTION: Part 1 - Step 6: Digest &amp; Clear Sample (optional) (buffer and enzyme may vary depending on sample type)
23. Incubate sample in 3 mL with 10 % at 20.000 U/mL for 180 min at 37 °C. 

SECTION: Part 1 - Step 7: Hybridize Encoding Probes
28. Wash and equilibrate sample by immersing slip-mounted sample in 41 °C pre-heated 200-500 µL for 30 min. 

SECTION: Part 1 - Step 7: Hybridize Encoding Probes
29. 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).
SECTION: Part 1 - Step 7: Hybridize Encoding Probes
30. Remove the slip from Wash Buffer A and carefully wipe away the excess buffer surrounding the sample.
SECTION: Part 1 - Step 7: Hybridize Encoding Probes
31. Dispense 125 µLcontaining 5 micromolar (µM) -200 micromolar (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.

SECTION: Part 1 - Step 7: Hybridize Encoding Probes
32. Incubate at 41 °C in a humidified chamber for 1080 min -1440 min  up to 2160 min .

SECTION: Part 1 - Step 8: Wash Away Residual Encoding Probes
33. Remove the hybridization buffer and carefully remove the excess buffer surrounding the sample.
SECTION: Part 1 - Step 8: Wash Away Residual Encoding Probes
34. Wash the sample in pre-heated 41 °C  Wash Buffer A for 30 min, two times.

SECTION: Part 1 - Step 8: Wash Away Residual Encoding Probes
35. Wash two times in 41 °C  pre-heated Encoding Wash Buffer (SSC-tw) for 5 min.

SECTION: Part 1 - Step 8: Wash Away Residual Encoding Probes
36. Wash two times in Room temperature  1x PBS.
SECTION: Part 1 - Step 9: Hybridize amplifiers (optional)
37. To label the gel embedded and cleared samples with primary and secondary amplifiers.
SECTION: Part 1 - Step 9: Hybridize amplifiers (optional)
38. Incubate sample in Wash Buffer C at 37 °C for 30 min. 

SECTION: Part 1 - Step 9: Hybridize amplifiers (optional)
39. Aspirate to remove Wash Buffer C.
SECTION: Part 1 - Step 9: Hybridize amplifiers (optional)
40. Hybridize primary amplifier.  Incubate the sample in a  125 µL of 5 nanomolar (nM)* in amplifier hybridization buffer for  15 min** 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.
SECTION: Part 1 - Step 9: Hybridize amplifiers (optional)
41. Wash 3 times with Wash C for 5-10 min each at Room temperature. 

SECTION: Part 1 - Step 9: Hybridize amplifiers (optional)
42. Hybridize secondary amplifier.  Incubate the sample in a  125 µL 5 nanomolar (nM)* in amplifier hybridization buffer for  15 min** 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.

SECTION: Part 1 - Step 9: Hybridize amplifiers (optional)
43. Wash twice in Room temperature Wash C for 5 min each followed by a 15 min  - 30 min wash in 37 °C  Wash C.

SECTION: Part 1 - Step 9: Hybridize amplifiers (optional)
44. Perform MULTIPLEXED ITERATIVE FISH Imaging (Part 2) immediately or store sample for up to 24 hours in storage buffer at 4 °C. 

SECTION: Part 2a - MULTIPLEXED ITERATIVE FISH Imaging with Fluidics System
45. 
 

SECTION: Part 2a - MULTIPLEXED ITERATIVE FISH Imaging with Fluidics System
46. 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.
SECTION: Part 2a - Step 1: Prepare Solutions for Imaging
47. 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
SECTION: Part 2a - Step 2: Assemble Fluidics System
48. 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.
SECTION: Part 2a - Step 2: Assemble Fluidics System
49. Ensure the system is fully assembled, plugged in and turned on.
SECTION: Part 2a - Step 2: Assemble Fluidics System
50. Double-check correctness of the details for the pump protocol for the MULTIPLEXED ITERATIVE FISH
Fluidics for the current project.
SECTION: Part 2a - Step 2: Assemble Fluidics System
51. Load the sample to the flow cell and connect.
SECTION: Part 2a - Step 2: Assemble Fluidics System
52. Carefully load all solutions to the proper reservoirs.
SECTION: Part 2a - Step 3: MULTIPLEXED ITERATIVE FISH Imaging Protocol
53. 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:
SECTION: Part 2a - Step 3: MULTIPLEXED ITERATIVE FISH Imaging Protocol
54. Readout hybridization buffer (with readout probes)

a.    Run 2 mL over 3 min to prime buffer to the sample.
b.    Run additional 2.5 mL over the sample for 4 min.
c.    Pause flow for 15 min - 120 min depending on sample size 
            (10 µm = 15 min, 30 µm = 60 min, 100 µm = 120 min).

SECTION: Part 2a - Step 3: MULTIPLEXED ITERATIVE FISH Imaging Protocol
55. Readout Wash Buffer (Wash D)
   a. Run   2 mL  over 210 s to flush.
   b. Run  2 mL  over 10 min to wash.

SECTION: Part 2a - Step 3: MULTIPLEXED ITERATIVE FISH Imaging Protocol
58. Imaging. Pause fluidics and proceed with imaging.
SECTION: Part 2a - Step 3: MULTIPLEXED ITERATIVE FISH Imaging Protocol
59. TCEP Cleavage Buffer - 
a. Run 2 mL  in 210 s min to prime cleavage buffer to the sample.
b. Run 1 mL  over sample for  5 min
c.Pause flow for 10 min 

SECTION: Part 2a - Step 3: MULTIPLEXED ITERATIVE FISH Imaging Protocol
60. 2x SSC Wash Buffer (Wash B) -
a. Run  2 mL  in 10 min to run cleavage buffer over sample and prime SSC buffer.
b. Run  2 mL  in 4 min to rinse off cleavage buffer. 

SECTION: Part 2a - Step 3: MULTIPLEXED ITERATIVE FISH Imaging Protocol
61. Repeat steps 54-60 for each readout round.
SECTION: Part 2a - Step 3: MULTIPLEXED ITERATIVE FISH Imaging Protocol
62. When all readout rounds are complete proceed with step 63.
SECTION: Part 2a - Step 3: MULTIPLEXED ITERATIVE FISH Imaging Protocol
63. DAPI Stain 

a. Run 2 mL  DAPI in 2xSSC (Wash B) for 60 min.
    i.    Use 50 µg/mL DAPI for thick (40 μm) samples.
    ii.    Use 1 µg/mL -10 µg/mL DAPI for 10 μm samples.

SECTION: Part 2a - Step 3: MULTIPLEXED ITERATIVE FISH Imaging Protocol
64. 2xSSC (Wash B) - 2 mL for 210 s to flush.
SECTION: Part 2a - Step 3: MULTIPLEXED ITERATIVE FISH Imaging Protocol
65. Imaging Buffer - 2 mL  in 6 min  then halt flow.
SECTION: Part 2a - Step 3: MULTIPLEXED ITERATIVE FISH Imaging Protocol
66. Proceed with Imaging. 
SECTION: Part 2b - Alternate MULTIPLEXED ITERATIVE FISH Imaging without Fluidics
67. 
 

SECTION: Part 2b - Alternate MULTIPLEXED ITERATIVE FISH Imaging without Fluidics
68. 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.
SECTION: Part 2b - Step 1a: MULTIPLEXED ITERATIVE FISH Imaging Protocol - Single Hybridization using coverslip mounted sample.
69. Readout Probe Hybridization.

a.    Pipette 200 µL in Readout Hybridization Buffer to sample and incubate at Room temperature  for 10 min.
b.    Aspirate Readout Hybridization Buffer from the sample.

SECTION: Part 2b - Step 1a: MULTIPLEXED ITERATIVE FISH Imaging Protocol - Single Hybridization using coverslip mounted sample.
70. Wash away unbound probe by adding 200 µL  to sample for 5 min, two times. Additional washes may improve the result.
SECTION: Part 2b - Step 1a: MULTIPLEXED ITERATIVE FISH Imaging Protocol - Single Hybridization using coverslip mounted sample.
71. Dapi Stain. Add 200 µL  with DAPI nuclear stain (at 1μg/mL) to sample and incubate for 30 min  at 37 °C .

SECTION: Part 2b - Step 1a: MULTIPLEXED ITERATIVE FISH Imaging Protocol - Single Hybridization using coverslip mounted sample.
72. Remove the Dapi stain and wash with Wash Buffer B for 5 min, two times.
SECTION: Part 2b - Step 1a: MULTIPLEXED ITERATIVE FISH Imaging Protocol - Single Hybridization using coverslip mounted sample.
73. Add 100 µL -200 µL to sample and mount to glass plate with clear nail polish.

SECTION: Part 2b - Step 1a: MULTIPLEXED ITERATIVE FISH Imaging Protocol - Single Hybridization using coverslip mounted sample.
74. Proceed with imaging.
SECTION: Part 2b - Step 1b: MULTIPLEXED ITERATIVE FISH Imaging - Multiple Hybridizations Using Chamber-slip
75. Readout Probe Hybridization.

a.    Pipette 200 µL in Readout Hybridization Buffer to sample and incubate at Room temperature  for 10 min.
b.    Aspirate Readout Hybridization Buffer from the chambers.
SECTION: Part 2b - Step 1b: MULTIPLEXED ITERATIVE FISH Imaging - Multiple Hybridizations Using Chamber-slip
76. Wash away unbound probe by adding 200 µL  to sample for 5 min, two times. Additional washes may improve the result.
SECTION: Part 2b - Step 1b: MULTIPLEXED ITERATIVE FISH Imaging - Multiple Hybridizations Using Chamber-slip
77. Add 100 µL -200 µL to sample. 
SECTION: Part 2b - Step 1b: MULTIPLEXED ITERATIVE FISH Imaging - Multiple Hybridizations Using Chamber-slip
79. TCEP Cleavage Buffer – 100 µL for 15 min.

SECTION: Part 2b - Step 1b: MULTIPLEXED ITERATIVE FISH Imaging - Multiple Hybridizations Using Chamber-slip
80. 2x SSC Wash Buffer (Wash B) – 250 µL each well, three times.

SECTION: Part 2b - Step 1b: MULTIPLEXED ITERATIVE FISH Imaging - Multiple Hybridizations Using Chamber-slip
81. Repeat steps 81-85 for each probe set round.
SECTION: Part 2b - Step 1b: MULTIPLEXED ITERATIVE FISH Imaging - Multiple Hybridizations Using Chamber-slip
82. Move on to step 88 when all rounds are complete.
SECTION: Part 2b - Step 2: Dapi Stain the Sample
83. Add 200 µL  with DAPI nuclear stain (at 1μg/mL) to sample and incubate for 30 min  at 37 °C .
SECTION: Part 2b - Step 2: Dapi Stain the Sample
84. Wash sample in Wash Buffer B for 5 min two times.
SECTION: Part 2b - Step 3:
85. Proceed to Imaging of the Sample.
SECTION: Part 1 - Step 2: Mount, SDS Pretreat and Permeabilize Sample
7. 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.
SECTION: Part 1 - Step 2: Mount, SDS Pretreat and Permeabilize Sample
6. 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 5 min at 4 °C (recommended) in a Pyrex 60mm petri dish (Fisher 08-747A). (For faster results, sample can be incubated in EtOH for 60 min at Room temperature).

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

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

iv. Aspirate rinse from the sample and let dry.

SECTION: Part 1 - Step 6: Digest &amp; Clear Sample (optional) (buffer and enzyme may vary depending on sample type)
22. Note: For lung tissue start at step 22.  Skip ahead to step 24 for brain tissue.
SECTION: Part 2a - Step 3: MULTIPLEXED ITERATIVE FISH Imaging Protocol
57. 1. 2x SSC Wash Buffer (Wash B)
    a. Run 1 mL  over 3 min to move 1mL imaging buffer over the sample.  
SECTION: Part 2a - Step 3: MULTIPLEXED ITERATIVE FISH Imaging Protocol
56. Imaging Buffer
a.  Run  2 mL  over 10 min to run remaining wash D over sample and prime imaging buffer to the sample.
67. 
SECTION: Document Summary
1. 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.
SECTION: Part 2b - Step 1b: MULTIPLEXED ITERATIVE FISH Imaging - Multiple Hybridizations Using Chamber-slip
78. Proceed with imaging of the round.