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 .
