Expansion-assisted iterative fluorescence in situ hybridization (EASI-FISH) in Drosophila CNS
Mark Eddison, Gudrun Ihrke
Abstract
A straightforward, robust, and reliable protocol (EASI-FISH) that utilizes expansion microscopy and the hybridization chain reaction for multiplexed in situ hybridization in thick slices of the mouse brain has recently been described (Wang et al., 2021). Below details a modified version of the EASI-FISH protocol for adult Drosophila CNS, which includes antibody detection of fluorescent reporters. The protocol also works well for larval CNS and is expected to be applicable to other tissue types.
Attachments
Steps
Fly brain dissection
Dissect fly CNS in S2 medium.
(for more details see attached protocol)
Fix up to 20 brains or 10 CNS in 2mL of 2% PFA/S2 medium for 0h 55m 0s in the dark on a nutator.
Rinse sample 1 x 2mL PBST (0.5% Triton).
Wash sample 4 x 0h 15m 0s 1mL PBST(0.5%Triton) on a nutator.
Rinse sample 1 x 2mL 70% EtOH.
Store brains in 2mL 70% EtOH @ 4°C for up to 6 months.
Day 1: Labelling RNA
Transfer brains to a 0.2ml PCR tube (2-4 brains per tube).
Rehydrate with 2 x 5 min wash in 150µL PBT (0.1%).
Rehydrate with 2 x 0h 5m 0s wash in 150µL PBT (0.1%) (1/2).
Rehydrate with 2 x 0h 5m 0s wash in 150µL PBT (0.1%) (2/2).
Incubate brains 1 x 0h 30m 0s in 150µL 20millimolar (mM) MOPS Buffer.
Thaw Melphalan-X (MelphX) and Acryloyl-X (Ac-X) solutions.
Using a P20 pipette, take off as much MOPS buffer from the brains as possible.
Dilute Melphalan-X stock 1:1 with MOPS Buffer (to 1mg/ml).
Add 1/100 Ac-X (10mg/mL) to Melphalan-X working solution. Vortex, mix, and spin.
Add 30µL of Melphalan-X/AcX solution to each PCR tube and gently mix.
Incubate 0h 2m 0s @ 37°C.
Prepare gel chambers for gelation the next day. Wipe a non-charged slide with RNase away. Adhere a gasket (4-6 wells max) and coat the glass surface of the chamber with 1µL poly-Lysine using a P20 pipette tip. Air dry and repeat.
Day 2: Gelation and Proteinase K digestion
Wash brains 2 x 2 min 150µL PBT and 1 x 2 mins 150µL PBS.
Wash brains 2 x 0h 2m 0s 150µL PBT and 1 x 0h 2m 0s 150µL PBS.
Wash brains 2 x 0h 2m 0s 150µL PBT and 1 x 0h 2m 0s 150µL PBS.
Thaw Stock-X and 4HT, Temed and APS. Vortex well and keep On ice.
Gently stick down brains in the center of the chamber, once stuck down, carefully add a drop of PBS to prevent dehydration.
Mix together Stock-X and 4HT, Temed and APS at a ratio of 94:2:2:2. Vortex.
Remove PBS from chamber with pipette tip and carefully wick away remaining PBS with a tissue.
Pipette 40µL of gel solution, on top of the brain, to each chamber. Incubate slide in the fridge ( 4°C ) for 0h 10m 0s.
Take off gel solution and repeat step 22.
Take off the gel solution and gasket surface adhesive. Add a final 40µL of gel solution and gently place a cover slip over the chamber. Gently press to seal the coverslip and incubate @ 4°C for 0h 10m 0s.
Polymerize the gel @ 37°C for 1h 30m 0s to 2h 0m 0s.
Cool gels on the bench for a few minutes.
Take off the chamber lid and gasket with a razor blade. Trim the gels into a rectangle and nick the top right-hand corner to track the orientation of the sample.
Take off the gel from the slide with a paintbrush that has been wetted with a small amount of ProK Buffer and transfer it to a 2mL Eppendorf.
Incubate each gel with 1mL ProK Buffer and 10µL (1/100) ProK Enzyme @ 37°C 2h 0m 0s.
Day 3: Hybridization
Wash gels 4 x 15 min with 1mL PBS at Room temperature.
Wash gels 4 x 0h 15m 0s in 1mL PBS at Room temperature (1/4).
Wash gels 4 x 0h 15m 0s in 1mL PBS at Room temperature (2/4).
Wash gels 4 x 0h 15m 0s in 1mL PBS at Room temperature (3/4).
Wash gels 4 x 0h 15m 0s in 1mL PBS at Room temperature (4/4).
DAPI stain gels for 0h 10m 0s with 1mL DAPI/PBS (500 ng/ml).
Rinse with PBS.
Use a dissection scope with a UV bulb to neatly trim gel edges with a razor blade.
Thaw and mix hybridization (hyb) and probe wash buffer. Make sure hyb buffer is clear.
Incubate gel in 500µL hyb buffer for 0h 30m 0s @ 37°C .
Dilute probes 1/100 (10Nanomolar (nM)), in 300µL hyb buffer per gel. Vortex. Incubate @ 37°C.
Incubate gels with probes overnight @ 37°C, no shaking necessary.
Put probe wash buffer and PBS @ 37°C.
Day 4: Probe Washing
Wash 3 x 30 min 750µL Probe Wash Buffer @ 37°C.
Wash 3 x 0h 30m 0s 750µL Probe Wash Buffer @ 37°C (1/3).
Wash 3 x 0h 30m 0s 750µL Probe Wash Buffer @ 37°C (2/3).
Wash 3 x 0h 30m 0s 750µL Probe Wash Buffer @ 37°C (3/3).
Wash 3 x 30 min 1mL PBS @ 37°C.
Wash 3 x 0h 30m 0s 1mL PBS @ 37°C (1/3).
Wash 3 x 0h 30m 0s 1mL PBS @ 37°C (2/3).
Wash 3 x 0h 30m 0s 1mL PBS @ 37°C (3/3).
Wash 3 x 1hr 1mL PBS @ 37°C.
Wash 3 x 1h 0m 0s 1mL PBS @ 37°C (1/3).
Wash 3 x 1h 0m 0s 1mL PBS @ 37°C (2/3).
Wash 3 x 1h 0m 0s 1mL PBS @ 37°C (3/3).
Keep gels in PBS at Room temperature 1h 0m 0s.
Day 5: Hybridization Chain Reaction (HCR)
Incubate gels in 500µL Amplification buffer for at least 0h 30m 0s @ Room temperature
Snap cool hairpins with PCR machine @ 95°C for 0h 1m 30s and cool @ Room temperature for 0h 30m 0s.
Mix hairpins h1 and h2 @ 1/100 in 300µL Amp Buffer per gel. Vortex.
Incubate gel with hairpins for 3h 0m 0s @ Room temperature in the dark.
Wash gels 2 x 20 min in 750µL 5X SSCT @ Room temperature.
Wash gels 2 x 0h 20m 0s in 750µL 5X SSCT @ Room temperature (1/2).
Wash gels 2 x 0h 20m 0s in 750µL 5X SSCT @ Room temperature (2/2).
Wash gels 2 x 40 min in 1mL 0.5X SSCT @ Room temperature.
Wash gels 2 x 0h 40m 0s in 1mL 0.5X SSCT @ Room temperature (1/2).
Wash gels 2 x 0h 40m 0s in 1mL 0.5X SSCT @ Room temperature (2/2).
Stain sample with 500µL of anti-GFP-488 Ab @ (1/500) in PBT (0.1%) containing 5mg/mL Ultrapure BSA and incubate 0h 15m 0s (or the weekend) @ 4°C .
Day 6: Mount and Image
Wash 2 x 30 min with 1mL PBS-Triton (0.1%).
Wash 2 x 0h 30m 0s with 1mL PBS-Triton (0.1%) (1/2).
Wash 2 x 0h 30m 0s with 1mL PBS-Triton (0.1%) (2/2).
Wash 2 x 30 min and 1 x 1hr with 1mL PBS.
Wash 2 x 0h 30m 0s with 1mL PBS (1/2).
Wash 1 x 1h 0m 0s with 1mL PBS (2/2).
DAPI stain gels for 0h 15m 0s with 1mL PBS/DAPI (500ng/ml).
Mount gels (sample up) on an 8mm poly-lysine coated coverslip superglued to a Z.1 light-sheet sample holder and image.
For gel removal from the holder, incubate gels in 750µL of 10% Dextran Sulphate for 20 mins. Gels will shrink and fall off the coverslip.
For long-term storage, keep gels in 750µL 10% Dextran Sulphate @ 4°C
Stripping Probes and Hairpins for Multiplexing
Incubate gel for 0h 30m 0s in 1mL of DNAse1 Buffer @ 37°C.
Add 450µL of DNase Buffer to 50µL DNase1. Mix.
Incubate gel in DNase1 for 2h 0m 0s @ 37°C.
Wash 4 x 15 min with 1mL PBS.
Wash 4 x 0h 15m 0s with 1mL PBS (1/4).
Wash 4 x 0h 15m 0s with 1mL PBS (2/4).
Wash 4 x 0h 15m 0s with 1mL PBS (3/4).
Wash 4 x 0h 15m 0s with 1mL PBS (4/4).
Hybridize with next round of probes (Day 3, step 34).
JF-669 conjugation to unlabelled hairpins
Turn on speed vacuum (eg. Thermofisher, SPD120) and defrost dye at Room temperature for 0h 30m 0s.
Resuspend 2mg of JF-669, SE in 630µL Acetonitrile, mix and vortex, and aliquot in 30µL into labelled skirted 0.5ml screw-cap centrifuge tubes > each will contain 0.1mg of dye.
Evaporate acetonitrile in speed vacuum (in organic solvent mode) for 0h 45m 0s. Can store @ -20°C.
In two 1.5ml Eppendorf tubes, evaporate 5µL (500picomolar (pM)/10µg) of unlabelled hairpins h1 and h2 using a speed vac, in aqueous mode, for 0h 30m 0s. If you have 10µL (1nanomolar (nM)) use two tubes per hairpin. Check they have been fully evaporated.
Add 3µL of 0.1Molarity (M) Sodium Bicarbonate pH 8-9 to each evaporated hairpin. Mix.
Add 2µL of anhydrous DMSO to 0.1mg of dye. Mix.
Add 2µL dye mix (100µg) to each 3µL of hairpin (10µg). Mix. It will change colour.
Leave hairpin-dye mixture to react at Room temperature in the dark.
The next morning, add 5µL nuclease-free water to bring to 10µL.
Remove excess dye with a QIAquick Nucleotide removal kit (add 100µL PN1).
Elute dye-oligo conjugate in 50µL nuclease-free water.
Check the hairpin concentration on a spectrophotometer (eg. a NanoDrop One) and dilute to 60ng/μl.
Separately store hairpins h1-669 and h2-669 in 25µL aliquot’s in a PCR tube@ -20°C.
Test conjugation by HCR.
