Multiplex FISH in CaRMA-imaged tissue after in vivo calcium imaging

Multiple rounds of FISH using RNAscope® multiplex detection reagents on FISH-only tissue (lacking GCaMP/not post-in vivo imaging), aimed to register across rounds 

Materials and reagents:

1. 14 µm-thick tissue sections mounted onto Fisherbrand™ Superfrost™ Plus microscope slide (Cat # 12-550-15), air dried in -4°C overnight after sectioning or retrieved from -80°C storage 
2. RNase Away (Thermo ScientificTM Cat # 7002)
3. DAPI (Millipore Sigma D9542, use 0.01 mg/ml for staining)
4. 4% paraformaldehyde (PFA)
5. RNase-free DNase I (Qiagen Cat # 79254, 2 K units/µl in the buffer (Qiagen buffer RDD) that comes with the kit, use 75-100 µl to cover the entire section)
   • It is critical to use the correct buffer, since DNase-I requires specific cation composition in the buffer. This buffer is also available separately as Qiagen Cat # 1011132)
6. Molecular biology grade water (abbreviated as “MBG water” below, CORNING REF46-000-CM)
7. 1X PBS (Fisher Bioreagents BP24384)
8. Vector Laboratories ImmEDGE hydrophobic barrier pen (Cat # H-4000)
9. Phenylmethylsulfonyl fluoride (PMSF, Millipore Sigma-Aldrich SKU 93482). Make 10 mM PMSF stock solution in EtOH. Dilute the stock solution with MBG water to a final working concentration of 1 mM 
10. 1 mM CuSO4 in 1× SSC
11. Yeast tRNA (Thermo Fisher Scientific Cat # AM7119), use at 10 mg/mL
12. RNAscope® reagents: 
    • Probes for genes of interest
    • Target retrieval reagents (Cat # 322000)
    • Protease IV (Cat # 322336) in the protease III and IV reagent kit (Cat # 322340)
    • Fluorescent multiplex detection reagents (Cat # 320851)
    • Wash buffer (Cat # 310091)

Day 1 procedures

1. Wipe down bench surfaces and instruments with 70% EtOH and then with RNase-Away to ensure a clean and RNase-free experimental environment. 
2. Let the tissue sections air dry in an aerated hood for 30 min at room temperature (RT).
3. Pipette DAPI solution (200-300 µl, depending on section size) onto each section to completely cover the section without overflowing the glass slide. Incubate for 10 min at RT.
4. Remove staining solution by tilting the slide and let the staining solution flow onto a paper towel. Wash the stain by pipetting MBG water to cover the section, and immediately tilt the slide to remove the water. Repeat this wash once more. (We call this wash “pipette-then-tilt wash” from here onward.)
5. Pipette 1X PBS to cover the section to restore tissue osmolarity. Then tilt the slide to remove PBS
6. Blot wet area of the glass slide around the section without touching the section. Let the slide air dry for 10 to 15 min, if necessary. 
7. Pipette 4% PFA to cover the entire section. Incubate at RT for 30 min. 
8. Remove PFA, Pipette-then-tilt wash with 1X PBS twice and MBG water twice.
9. Blot wet area of the glass slide around the section without touching the section. Let the slide air dry for 10 to 15 min, if necessary. 
   • Examine the tissue section at this step. It should be securely mounted onto the slide, flat without curling, wrinkling, corner lifting from the slide, etc. Any of these phenomena indicates compromised section quality. One might consider optimizing brain tissue harvesting, sectioning, and/or storage conditions. 
10. Pipette RNase-free DNase I to cover the entire section, incubate at 37°C in a moisture preserving environment (latch-locked chamber used for RNAScope® multiplex assay, a large petri dish with lid, etc.; chamber/ dish must be lined with wet paper towel.) for 4 hours. Exchange for fresh DNase-I solution after the first 2 hours. 
    • “Exchange” can be done by tilting the slide to let the current DNase-I incubation solution flow onto a paper towel, blotting the wet area around the section, and pipetting fresh DNase-I solution to cover the section.
    • DNase I digestion at this stage (after DAPI initial staining, paraformaldehyde fixing, but before FISH) allows to generate consistent DAPI staining patterns throughout all rounds of imaging.
    • When first setting up the experiment, examine the sample frequently during incubation to ensure the DNase-I solution is held by surface tension as a “dome” to cover the section throughout the entire 4-hour incubation time. 
    • Note, DNase-I requires divalent cations and is inhibited by monovalent cations. Thus, before applying DNase-I, the last solution that touches the section is usually MBG water, not 1X PBS. In contrast, when a DNase-I incubation terminates, immediately wash with 1X PBS, which inhibits DNase-I activity. 
11. Remove the DNase-I solution, Pipette-then-tilt wash the section with 1X PBS 3 times. Blot wet area of the glass slide around the section without touching the section. Let the slide air dry for 15 - 20 minutes.
    • Section should appear dry but not dehydrated. Signs of dehydration include wrinkles/cracks within the tissue, visible salt granule deposits onto of the section, etc.
    • Examine the tissue section at this step. It should be securely mounted onto the slide, flat without curling, wrinkling, corner lifting from the slide, etc. Any of these phenomena indicates compromised section quality. 
12. Perform the target retrieval step according to RNAscope® protocol with the following modification: after brining the target retrieval solution to a boil, monitor and maintain the solution between 90 and 95 °C with a thermometer, and incubate the slide in the temperature-monitored target retrieval solution for 45 sec.
13. Let the slide and section dry completely in RT. Draw hydrophobic barrier on the glass slide around the section. Let the barrier dry completely at RT. 
14. Perform protease IV treatment according to RNAscope® protocol with the following modification: extend protease IV digestion to 105 min and inactivate residual protease IV activity by incubating the sections with 1 mM PMSF at RT for 30 min. 
15. Tilt the slide to remove PMSF, Pipette-then-tilt wash the section once with MBG water, then twice with 1X PBS. 
16. Pipette 1X PBS into the hydrophobic barrier to cover the tissue section and store in 4°C overnight. 

Day 2 procedures

1. Retrieve the store slides, examine, and ensure the 1X PBS within the hydrophobic barrier have securely covered the section during overnight storage (no PBS overflow out of the hydrophobic barrier, etc.)
2. Tilt the slide to remove 1X PBS, incubate the section with 1 mM CuSO₄ (in 1X SSC) for 30 min at RT.
3. Tilt the slide to remove CuSO_4. Pipette-then-tilt wash the section with MBG water once then with 1X PBS twice. 
4. Incubate the section with 10 mg/mL yeast tRNA at 40°C for 30 min in a moisture-preserving environment (e.g., latch-locked chamber used for RNAScope® multiplex assay). Tilt the slide to remove yeast tRNA solution. Pipette-then-tilt wash the section twice with 1X PBS. 
5. Carry out RNAScope® Multiplex Assay according to the manufacture’s protocol. Do NOT cover-slip the section. Rather, image DAPI and FISH signals on a confocal microscope with water-dipping objective and 1X PBS as the immersion media. 
   • Note that in post hoc FISH experiments on GCaMP6m-expressing mouse brain tissue, we used SSC buffered citric acid as the immersion media. The acidic media was formulated to quench GCaMP6m fluorescence. 
6. Pipette-and-tilt wash the section with MBG water 3 times. Then Repeat “Day 1 Step 10” to strip signal amplification oligonucleotides and probes from the 1^st round of FISH
7. Remove the DNase-I solution, Pipette-then-tilt wash the section with 1X PBS 3 times 
   • When a user sets up the assay for the first time, we recommend performing a 4-min wash in the RNAScope multiplex assay wash buffer at this stage and examining the stripping results under the same confocal microscope settings used for FISH imaging. 
   • Once FISH signals are removed, one should perform the following check: omit the RNAScope probing step but repeat all the amplification steps. The absence of FISH signal confirms that stripping has removed not only amplification oligonucleotides but also gene-specific probes.
8. Store the section as described in “Day 1 Step 16” for the next round of FISH. 
9. Repeat Day 2 procedure 3 times to reach 4 rounds of FISH. 
10. Pipette-then-tilt wash the section 3 times with 1X PBS, let the slide dry in an aerated hood, and store it in -80°C for future reference/re-examination.