TITLE: Multiplexed Iterative FISH Experimental Protocol SOP002.v3.5
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) 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.  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]
v3.5 revision notes
Minor edits and typo corrections.
Updated solution preparations with manufacturer and catalog numbers as well as adding Amplifier and Saber Encoding Hybridization Buffer master mixes.
Updated linker hybridization and wash temperatures from 41°C to 37°C.
Added adjustment note for amplifier hybridizations.

[STEPS]
SECTION: Part 1 - Tissue or Cell-Based Experiment Preparation
1. 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 for 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 - Tissue or Cell-Based Experiment Preparation
2. 
 

SECTION: Part 1 - Step 1: Coverslip Functionalization
3. Refer to current version of SOP003 for protocol on Coverslip Functionalization. 
SECTION: Part 1 - Step 2: Mount, SDS Pretreat and Permeabilize Sample
9. To permeabilize the tissue, immerse the slip mounted tissue in 70 % (v/v)   
at 4 °C  (recommended) in a Pyrex 60 mm petri dish (Fisher 08-747A).
 


SECTION: Part 1 - Step 2: Mount, SDS Pretreat and Permeabilize Sample
10. Remove sample from ethanol, carefully wipe away excess ethanol surrounding tissue and let dry.
SECTION: Part 1 - Step 2: Mount, SDS Pretreat and Permeabilize Sample
11. Using 4 % grown on coverslip (optionally, use 8-well chamber slip).

SECTION: Part 1 - Step 2: Mount, SDS Pretreat and Permeabilize Sample
12. To permeabilize cells, immerse the slip mounted sample in 70 % (v/v)   
at 4 °C  (recommended) in a Pyrex 60 mm petri dish (Fisher 08-747A).
 


SECTION: Part 1 - Step 2: Mount, SDS Pretreat and Permeabilize Sample
13. Alternatively, pipette 100 µL  to each well and incubate at Room temperature  for 10 min with gentle rocking.

SECTION: Part 1 - Step 3: Hybridize Linker (optional; use when gel embedding and digesting sample)
16. 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)
17. 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)
18. Dispense 200 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.
SECTION: Part 1 - Step 3: Hybridize Linker (optional; use when gel embedding and digesting sample)
19. 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
20. Remove the hybridization buffer and carefully remove excess buffer surrounding sample.
SECTION: Part 1 - Step 4: Wash Away Residual Linker
21. Immerse slip in pre-heated 37 °C Wash Buffer A for 30 min , two times.

SECTION: Part 1 - Step 4: Wash Away Residual Linker
22. 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
23. Wash two times in Room temperature  1x PBS.

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

SECTION: Part 1 - Step 5: Gel Embed Sample (optional - gel embed when clearing and digesting)
25. 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)
26. Cast a thin PA film by adding 50 µL  to the sample and invert a smaller (25 mm) gel-slick coated coverslip onto the gel solutions being careful to avoid air bubbles. You may need to adjust the volume depending on the sample thickness.  Aim to cast a gel that is approximately the same thickness of your sample.

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

SECTION: Part 1 - Step 5: Gel Embed Sample (optional - gel embed when clearing and digesting)
28. After casting, carefully remove the smaller coverslip from your sample. If the coverslip is stuck, you can loosen the coverslip by immersing in Wash C or SDS-CS at 37 °C , depending on the next step for your experiment.

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

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

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

SECTION: Part 1 - Step 6: Clear &amp; Digest Sample (optional) (buffer and enzyme may vary depending on sample type)
32. Wash in PBS 3 times for 5 min each at Room temperature. 

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

SECTION: Part 1 - Step 6: Clear &amp; Digest Sample (optional) (buffer and enzyme may vary depending on sample type)
34. Wash the sample in 1x PBS 3 times for 5 min each at Room temperature.

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

SECTION: Part 1 - Step 7: Hybridize Encoding Probes
36. 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
37. 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
38. Dispense 200 µL containing 5 Micromolar (µM) -200 Micromolar (µM)  (depending on the number of unique encoding probes in the probe set) 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
39. 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
40. Remove the hybridization buffer and carefully remove the excess buffer surrounding the sample.
SECTION: Part 1 - Step 8: Wash Away Residual Encoding Probes
41. Immerse the slip in pre-heated 41 °C  Wash Buffer A for 30 min, two times.

SECTION: Part 1 - Step 8: Wash Away Residual Encoding Probes
42. Wash two times in 41 °C  pre-heated Encoding Wash Buffer (SSC-tw) for 5 mins.
SECTION: Part 1 - Step 8: Wash Away Residual Encoding Probes
43. Wash two times in Room temperature  1x PBS.
SECTION: Part 1 - Step 9: Hybridize amplifiers (optional)
44. To label the gel embedded and cleared samples with primary and secondary amplifiers.
SECTION: Part 1 - Step 9: Hybridize amplifiers (optional)
45. Incubate sample in Wash Buffer C at 37 °C for 30 min. 

SECTION: Part 1 - Step 9: Hybridize amplifiers (optional)
46. Remove from Wash Buffer C and blot off extra buffer.
SECTION: Part 1 - Step 9: Hybridize amplifiers (optional)
47. Hybridize primary amplifier. Invert sample onto a 50 µL of 5 Nanomolar (nM) in amplifier hybridization buffer for 15 min in humidity-controlled 37 °C incubator. Adjust amplifier hybridization volume and hybridization time according to sample size and thickness.

SECTION: Part 1 - Step 9: Hybridize amplifiers (optional)
48. Wash 3 times with Wash C for 5 min each at Room temperature. 

SECTION: Part 1 - Step 9: Hybridize amplifiers (optional)
49. Hybridize secondary amplifier. Invert sample onto a 50 µL of 5 Nanomolar (nM) in amplifier hybridization buffer for 15 min in humidity-controlled 37 °C incubator. Adjust amplifier hybridization volume and hybridization time according to sample size and thickness.

SECTION: Part 1 - Step 9: Hybridize amplifiers (optional)
50. Wash twice in Wash C for 5 min followed by a 15 min  wash in Wash C.

SECTION: Part 1 - Step 9: Hybridize amplifiers (optional)
51. 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
52. 
 

SECTION: Part 2a - MULTIPLEXED ITERATIVE FISH Imaging with Fluidics System
53. The basic imaging process for MULTIPLEXED ITERATIVE FISH involves iterative fluid exchange to apply dye-conjugated readout probes, washes, anti-photobleaching imaging buffer, reductive cleavage buffer and rinse.  A single round of fluidics takes approximately 45 minutes with an automated fluidics system not including imaging times.  We strongly suggest using an automated fluidics system to improve the consistency of each round as well as efficiency.  While manually controlling the fluidics system is possible, this would require inconvenient around the clock staffing to run a single multiple round experiment of 16 genes with two color readouts.
SECTION: Part 2a - Step 1: Prepare Solutions for Imaging
54. Prepare the following solutions with the corresponding volumes:

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
SECTION: Part 2a - Step 2: Assemble Fluidics System
55. Make sure that all tubing is properly connected. MULTIPLEXED ITERATIVE FISH probes and preparation
time are very cost intensive so leaks need to be avoided at all costs.
SECTION: Part 2a - Step 2: Assemble Fluidics System
56. Ensure the system is fully assembled, plugged in and turned on.
SECTION: Part 2a - Step 2: Assemble Fluidics System
57. 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
58. Load the sample to the fluidics chamber.
SECTION: Part 2a - Step 2: Assemble Fluidics System
59. Carefully load all solutions to the proper reservoirs.
SECTION: Part 2a - Step 3: MULTIPLEXED ITERATIVE FISH Imaging Protocol
60. 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 (example pump speeds listed in bold below).
SECTION: Part 2a - Step 3: MULTIPLEXED ITERATIVE FISH Imaging Protocol
61. (9) Readout hybridization buffer (with probes)

a.    Wash 2 mL over 5 min to flush.
b.    Wash additional 2 mL over sample in 6 min.

SECTION: Part 2a - Step 3: MULTIPLEXED ITERATIVE FISH Imaging Protocol
62. (4) Readout Wash Buffer (Wash D) - 2 mL  in 9 min.

SECTION: Part 2a - Step 3: MULTIPLEXED ITERATIVE FISH Imaging Protocol
63. (7) Imaging Buffer - 2 mL  in 6 min  then halt flow.

SECTION: Part 2a - Step 3: MULTIPLEXED ITERATIVE FISH Imaging Protocol
64. Imaging. Proceed with Imaging.
SECTION: Part 2a - Step 3: MULTIPLEXED ITERATIVE FISH Imaging Protocol
65. (3) TCEP Cleavage Buffer - 3 mL  in 15 min.

SECTION: Part 2a - Step 3: MULTIPLEXED ITERATIVE FISH Imaging Protocol
66. (10) 2x SSC Wash Buffer (Wash B) - 2 mL  in 4 min.

SECTION: Part 2a - Step 3: MULTIPLEXED ITERATIVE FISH Imaging Protocol
67. Repeat steps 63-68 for each probe set.
SECTION: Part 2a - Step 3: MULTIPLEXED ITERATIVE FISH Imaging Protocol
68. When all readout rounds are complete proceed with steps 8-11.
SECTION: Part 2a - Step 3: MULTIPLEXED ITERATIVE FISH Imaging Protocol
69. DAPI Stain – Wash 2 mL  DAPI in 2xSSC (Wash B) for 10 min.

a.    Use 50 µg/mL for thick (40 μm) samples.
b.    Use 1 µg/mL -10 µg/mL for 10 μm samples.

 


SECTION: Part 2a - Step 3: MULTIPLEXED ITERATIVE FISH Imaging Protocol
70. 2xSSC (Wash B) - 2 mL for 4 min.

SECTION: Part 2a - Step 3: MULTIPLEXED ITERATIVE FISH Imaging Protocol
71. Imaging Buffer - 2 mL  in 6 min  then halt flow.
SECTION: Part 2a - Step 3: MULTIPLEXED ITERATIVE FISH Imaging Protocol
72. Imaging. Image at 405 nm.
SECTION: Part 2b - Alternate MULTIPLEXED ITERATIVE FISH Imaging without Fluidics
73. 
 

SECTION: Part 2b - Alternate MULTIPLEXED ITERATIVE FISH Imaging without Fluidics
74. 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.
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 sample.

SECTION: Part 2b - Step 1a: MULTIPLEXED ITERATIVE FISH Imaging Protocol - Single Hybridization using coverslip mounted sample.
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 1a: MULTIPLEXED ITERATIVE FISH Imaging Protocol - Single Hybridization using coverslip mounted sample.
77. 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.
78. 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.
79. 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.
80. Proceed with imaging.
SECTION: Part 2b - Step 1b: MULTIPLEXED ITERATIVE FISH Imaging - Multiple Hybridizations Using Chamber-slip
81. 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
82. 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
83. Add 100 µL -200 µL to sample. Proceed with imaging of the round.
SECTION: Part 2b - Step 1b: MULTIPLEXED ITERATIVE FISH Imaging - Multiple Hybridizations Using Chamber-slip
84. TCEP Cleavage Buffer – 100 µL for 15 min.

SECTION: Part 2b - Step 1b: MULTIPLEXED ITERATIVE FISH Imaging - Multiple Hybridizations Using Chamber-slip
85. 2x SSC Wash Buffer (Wash B) – 250μL each well, three times.
SECTION: Part 2b - Step 1b: MULTIPLEXED ITERATIVE FISH Imaging - Multiple Hybridizations Using Chamber-slip
86. Repeat steps 81-85 for each probe set round.
SECTION: Part 2b - Step 1b: MULTIPLEXED ITERATIVE FISH Imaging - Multiple Hybridizations Using Chamber-slip
87. Move on to step 88 when all rounds are complete.
SECTION: Part 2b - Step 2: Dapi Stain the Sample
88. 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
89. Wash sample in Wash Buffer B for 5 min two times.
SECTION: Part 2b - Step 3:
90. Proceed to Imaging of the Sample.
SECTION: Part 1 - Step 2: Mount, SDS Pretreat and Permeabilize Sample
8. Pretreat tissue by washing in 4 % (SDS-CS), for 5 min minutes, four times.

SECTION: Part 1 - Step 2: Mount, SDS Pretreat and Permeabilize Sample
7. Mount tissue slices (10-40µM sections typically) to functionalized (silanized) coverslip and dry in 37°C oven for 30mins.
SECTION: Part 1 - Step 2: Mount, SDS Pretreat and Permeabilize Sample
6. Wash tissue slices twice in nuclease-free 1xPBS for 2 min -5 min each.

SECTION: Part 1 - Step 2: Mount, SDS Pretreat and Permeabilize Sample
5. If frozen, thaw tissue to Room temperature .

SECTION: Part 1 - Step 2: Mount, SDS Pretreat and Permeabilize Sample
4. If using 4 % , follow step 5.  For 4 % (v/v) ,  skip below to step 11.

SECTION: Part 1 - Step 2: Mount, SDS Pretreat and Permeabilize Sample
14. Aspirate rinse from the wells and let dry.
SECTION: Part 1 - Step 3: Hybridize Linker (optional; use when gel embedding and digesting sample)
15. Wash & equilibrate sample by immersing slip-mounted sample in 37°C pre-heated 200µL Wash Buffer A for 30 min.