Hybridized Chain Reaction Fluorescent in situ Hybridization (HCR-FISH)
[1.1] Collect juvenile squid from hatching table (refer to colonization protocol as needed) and isolate into collection cups.
Colonize with desired V. fischeri strain.
[1.2] Anesthetize squid for 2 min by placing into 2% ethanol in filtered seawater.
[1.3] Under dissecting scope, split and peel back mantle on anterior side.
Then carefully pull back the funnel to expose the light organ.
[1.4] Place dissected squid into 4% paraformaldehyde (PFA) and incubate overnight at 4 °C on shaker to fix.
The best vessels to use are 1.5mL screw-cap vials, to prevent leaking at later wash steps.
Permeabilization by Proteinase KUse RNase-free equipment and solutions throughout the remainder of the protocol.
Perform all treatments, hybridizations, and washes in a 500-μL volume, on a rotator/shaker, unless otherwise noted.
[2.1] Wash each sample five times (5 min per wash) with Permeabilization Buffer at room temperature (RT).
[2.2] Treat with 0.01 mg/mL Proteinase K Permeabilization Buffer at RT for 15-20 minutes.
Do not place on shaker or rotator.
[2.3] Stop the proteinase K digestion with two washes of 2 mg/mL glycine in Permeabilization Buffer.
[2.4] Post-fix in Permeabilization Buffer with 4% PFA for 1 h at RT on the shaker.
[2.5] Wash with Permeabilization Buffer five times (5 min per wash).
Note: Permeabilized juveniles can be used immediately, or stored for no longer than 1 week at 4 °C in Permeabilization Buffer.
Pre-hybridization for Probes.
[3.1] Remove as much of the Permeabilization Buffer as possible from the sample.
Incubate sample in 500 μL of 50% Hyb Buffer at 65 °C for 30 min.
[3.2] Change sample into 500 μL of fresh 50% Hyb Buffer, and incubate at 65 °C for 2.5 h.
Note: Prevent drying during prolonged incubations.
When using petri plates, place them in a humidified chamber or seal the cover of the plate with a strip of parafilm.
Probe Hybridization.
To identify possible artifacts and confounding effects, the following alternate sample preparations should be performed:
Autofluorescence (AF) – Follow protocol but do not add probes (step 4) or hairpins (step 7).
Non-Specific Amplification of hairpins (NSA) – Sample incubated without probes (step 4) but with hairpins included.
Non-Specific Detection of targets (NSD) – This control is applicable only for (i) transgenic (non-endogenous) targets, where a wild-type sample missing the target transcript is treated using the same protocol, and with the test probes and hairpins; or (ii) non-ubiquitous endogenous target transcript, where the locus of expression is known beforehand, and for which surrounding tissue can give an estimate of NSD in the same sample after treatment.
[4.1] Mix 1 pmol of each probe in 500 μL of 50% Hyb buffer at 45 °C for 30 min (this step should be coordinated with step [3.2] so that they are completed at the same time).
[4.2] Remove the 50% Hyb buffer from [3.2], and add this probe solution to samples for overnight (16 h) incubation at 45 °C.
Probe Washes.
All solutions used here must be pre-warmed to 45 °C.
Probe solution is not reused, and hence discarded at the start of the washes.
[5.1] Wash samples in 500 μL Probe Wash Buffer for 15 min at 45 °C.
[5.2] Wash samples in 500 μL (75% of Probe Wash Buffer + 25% of 5X SSC) for 15 min at 45 °C.
[5.3] Wash samples in 500 μL (50% of Probe Wash Buffer + 50% of 5X SSC) for 15 min at 45 °C.
[5.4] Wash samples in 500 μL (25% of Probe Wash Buffer + 75% of 5X SSC) for 15 min at 45 °C.
[5.5] Wash samples 2 times, in 500 μL of 5X SSC for 15 min at 45 °C.
[5.6] Wash samples 2 times, in 500 μL of 5X SSC for 30 min at 45 °C.
Pre-hybridization for Hairpins [6.1] Incubate samples in 500 μL of DNA Amplification Buffer at RT for 30 min.
[6.2] Incubate samples in fresh 500 μL of DNA Amplification Buffer at RT for 30 min.
[6.3] Aliquot 6 pmol (for every 100 μL of DNA Amplification Buffer) of each hairpin in a separate PCR tube.
[6.4] Heat the hairpins to 95 °C for 90 sec (e.g., using a PCR machine/thermal cycler).
[6.5] Store the heated hairpins in the dark for 30 min at RT (keep hairpins unmixed).
[6.6] Prepare 100 μL of fresh DNA Amplification Buffer equilibrated at RT.
Note: (Steps [6.2] through [6.6] should be coordinated so that they are completed at the same time for all samples).
Hairpin Amplification.
[7.1] Mix all hairpins in 100 μL of pre-equilibrated DNA Amplification Buffer at RT.
(Final concentration of each hairpin is 60 nM.)
Note: The volume of this incubation can be scaled up if needed (i.e., for high-abundance squid transcripts); however, the hairpin concentration must be kept constant.
[7.2] Remove final wash solution from [6.2] and add the hairpin solution to the samples for an overnight (16 h) incubation at RT.
[7.3] Wrap the sample tubes (or incubation oven) in aluminum foil to keep light out.
Hairpin Washes.
Note: All solutions used here must be pre-equilibrated to RT.
[8.1] Wash samples 4 times, in 500 μL of 5X SSCTw for 5 min each at RT.
[8.2] Wash samples 2 times, in 500 μL of 5X SSCTw for 30 min each at RT.
Imaging.
[9.1] Samples can be imaged directly in 5X SSCTw, or stored in 5X SSCTw at 4 °C.
Note: The processed samples can be counterstained with phalloidin or wheat germ agglutinin following standard protocols.
