TITLE: Expansion-assisted iterative fluorescence in situ hybridization (EASI-FISH) in Drosophila CNS
AUTHORS: Mark Eddison, Gudrun Ihrke

[DESCRIPTION]
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. 


[GUIDELINES]

Reference: 

Wang et al., EASI-FISH for thick tissue defines lateral hypothalamus spatio-molecular organization. Cell184 (2021) 6361-6377.e24. 

https://pubmed.ncbi.nlm.nih.gov/34875226/



[STEPS]
SECTION: Fly brain dissection
1. 
Dissect fly CNS in S2 medium.
(for more details see attached protocol)


SECTION: Fly brain dissection
2. Fix up to 20 brains or 10 CNS in 2 mL of 2% PFA/S2 medium for 55 min in the dark on a nutator.

SECTION: Fly brain dissection
3. Rinse sample 1 x 2 mL PBST (0.5% Triton).

SECTION: Fly brain dissection
4. Wash sample 4 x 15 min 1 mL PBST(0.5%Triton) on a nutator.

SECTION: Fly brain dissection
5. Rinse sample 1 x 2 mL 70% EtOH.

SECTION: Fly brain dissection
6. Store brains in 2 mL 70% EtOH @ 4 °C for up to 6 months.

SECTION: Day 1: Labelling RNA
7. Transfer brains to a 0.2ml PCR tube (2-4 brains per tube).

SECTION: Day 1: Labelling RNA
8. Rehydrate with 2 x 5 min wash in 150 µL PBT (0.1%).

SECTION: Day 1: Labelling RNA
9. Incubate brains 1 x 30 min in 150 µL 20 millimolar (mM) MOPS Buffer.

SECTION: Day 1: Labelling RNA
10. Thaw Melphalan-X (MelphX) and Acryloyl-X (Ac-X) solutions.
SECTION: Day 1: Labelling RNA
11. Using a P20 pipette, take off as much MOPS buffer from the brains as possible.
SECTION: Day 1: Labelling RNA
12. Dilute Melphalan-X stock 1:1 with MOPS Buffer (to 1mg/ml).
SECTION: Day 1: Labelling RNA
13. Add 1/100 Ac-X (10 mg/mL) to Melphalan-X working solution.  Vortex, mix, and spin.

SECTION: Day 1: Labelling RNA
14.  Add 30 µL of Melphalan-X/AcX solution to each PCR tube and gently mix.

SECTION: Day 1: Labelling RNA
15.  Incubate 2 min @ 37 °C.

SECTION: Day 1: Labelling RNA
16. 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 1 µL poly-Lysine using a P20 pipette tip. Air dry and repeat. 
  

SECTION: Day 2: Gelation and Proteinase K digestion
17. Wash brains 2 x 2 min 150 µL PBT and 1 x 2 mins 150 µL PBS. 

SECTION: Day 2: Gelation and Proteinase K digestion
18. Thaw Stock-X and 4HT, Temed and APS. Vortex well and keep on ice.

SECTION: Day 2: Gelation and Proteinase K digestion
22.  Pipette 40 µL of gel solution, on top of the brain, to each chamber. Incubate slide in the fridge ( 4 °C ) for 10 min.

SECTION: Day 2: Gelation and Proteinase K digestion
23. Take off gel solution and repeat step 22.

 


SECTION: Day 2: Gelation and Proteinase K digestion
21. Remove PBS from chamber with pipette tip and carefully wick away remaining PBS with a tissue.
SECTION: Day 2: Gelation and Proteinase K digestion
20. Mix together Stock-X and 4HT, Temed and APS at a ratio of 94:2:2:2. Vortex.
 


SECTION: Day 2: Gelation and Proteinase K digestion
19. Gently stick down brains in the center of the chamber, once stuck down, carefully add a drop of PBS to prevent dehydration. 

 


SECTION: Day 2: Gelation and Proteinase K digestion
24. Take off the gel solution and gasket surface adhesive. Add a final 40 µL of gel solution and gently place a cover slip over the chamber. Gently press to seal the coverslip and incubate @ 4 °C for 10 min.

 

SECTION: Day 2: Gelation and Proteinase K digestion
25. Polymerize the gel @ 37 °C for 90 min  to  120 min.

SECTION: Day 2: Gelation and Proteinase K digestion
26. Cool gels on the bench for a few minutes.
SECTION: Day 2: Gelation and Proteinase K digestion
27. 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.
SECTION: Day 2: Gelation and Proteinase K digestion
28. 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 2 mL Eppendorf.

SECTION: Day 2: Gelation and Proteinase K digestion
29. Incubate each gel with 1 mL ProK Buffer and 10 µL (1/100) ProK Enzyme @ 37 °C 120 min.

SECTION: Day 3: Hybridization
30. 
Wash gels 4 x 15 min with 1 mL PBS at Room temperature.

SECTION: Day 3: Hybridization
31. DAPI stain gels for 10 min with 1 mL DAPI/PBS (500 ng/ml). 

SECTION: Day 3: Hybridization
32. Rinse with PBS.
SECTION: Day 3: Hybridization
33. Use a dissection scope with a UV bulb to neatly trim gel edges with a razor blade. 
SECTION: Day 3: Hybridization
34. Thaw and mix hybridization (hyb) and probe wash buffer. Make sure hyb buffer is clear. 
SECTION: Day 3: Hybridization
35. Incubate gel in 500 µL  hyb buffer for 30 min @ 37 °C .

SECTION: Day 3: Hybridization
36. Dilute probes 1/100 (10 Nanomolar (nM)), in 300 µL  hyb buffer per gel. Vortex. Incubate @ 37 °C.
SECTION: Day 3: Hybridization
37. Incubate gels with probes overnight @ 37 °C, no shaking necessary. 

SECTION: Day 3: Hybridization
38. Put probe wash buffer and PBS @ 37 °C.

SECTION: Day 4: Probe Washing
39. Wash 3 x 30 min 750 µL Probe Wash Buffer @ 37 °C.

SECTION: Day 4: Probe Washing
40. Wash 3 x 30 min 1 mL PBS @ 37 °C.

SECTION: Day 4: Probe Washing
41.  Wash 3 x 1hr 1 mL PBS @ 37 °C.

SECTION: Day 4: Probe Washing
42. Keep gels in PBS at Room temperature 60 min.

SECTION: Day 5: Hybridization Chain Reaction (HCR)
43. 
Incubate gels in 500 µL Amplification buffer for at least 30 min @  Room temperature

SECTION: Day 5: Hybridization Chain Reaction (HCR)
44. Snap cool hairpins with PCR machine @ 95 °C for 90 s and cool @ Room temperature for 30 min.

SECTION: Day 5: Hybridization Chain Reaction (HCR)
45.  Mix hairpins h1 and h2 @ 1/100 in 300 µL Amp Buffer per gel. Vortex.

SECTION: Day 5: Hybridization Chain Reaction (HCR)
46.  Incubate gel with hairpins for 180 min @ Room temperature in the dark.

SECTION: Day 5: Hybridization Chain Reaction (HCR)
47.  Wash gels 2 x 20 min in 750 µL 5X SSCT @ Room temperature.

SECTION: Day 5: Hybridization Chain Reaction (HCR)
48.  Wash gels 2 x 40 min in 1 mL 0.5X SSCT @ Room temperature.

SECTION: Day 5: Hybridization Chain Reaction (HCR)
49.  Stain sample with 500 µL of anti-GFP-488 Ab @ (1/500) in PBT (0.1%) containing  5 mg/mL Ultrapure BSA  and incubate  15 min (or the weekend) @ 4 °C . 

SECTION: Day 6: Mount and Image
50. 
Wash 2 x 30 min with 1 mL PBS-Triton (0.1%).

SECTION: Day 6: Mount and Image
51. Wash 2 x 30 min and 1 x 1hr with 1 mL PBS.

SECTION: Day 6: Mount and Image
52. 
 DAPI stain gels for 15 min with 1 mL  PBS/DAPI (500 ng/ml).

SECTION: Day 6: Mount and Image
53.  
Mount gels (sample up) on an 8mm poly-lysine coated coverslip superglued to a Z.1 light-sheet sample holder and image.
SECTION: Stripping Probes and Hairpins for Multiplexing
56. 
Incubate gel for 30 min in 1 mL of DNAse1 Buffer @ 37 °C.

SECTION: Stripping Probes and Hairpins for Multiplexing
57. Add 450 µL of DNase Buffer to 50 µL DNase1. Mix. 

SECTION: Stripping Probes and Hairpins for Multiplexing
58. Incubate gel in DNase1 for 120 min @ 37 °C. 

SECTION: Stripping Probes and Hairpins for Multiplexing
59. Wash 4 x 15 min with 1 mL PBS.

SECTION: Stripping Probes and Hairpins for Multiplexing
60. Hybridize with next round of probes (Day 3, step 34).
SECTION: JF-669 conjugation to unlabelled hairpins
61. 
 


Turn on speed vacuum (eg. Thermofisher, SPD120) and defrost dye at Room temperature for 30 min. 

SECTION: JF-669 conjugation to unlabelled hairpins
62. 
Resuspend 2 mg of JF-669, SE in 630 µL Acetonitrile, mix and vortex, and aliquot in 30 µL into labelled skirted 0.5ml screw-cap centrifuge tubes > each will contain 0.1 mg of dye. 

SECTION: JF-669 conjugation to unlabelled hairpins
63. Evaporate acetonitrile in speed vacuum (in organic solvent mode) for 45 min. Can store @ -20 °C. 

SECTION: JF-669 conjugation to unlabelled hairpins
64. In two 1.5ml Eppendorf tubes, evaporate 5 µL (500 picomolar (pM)/10 µg) of unlabelled hairpins h1 and h2 using a speed vac, in aqueous mode, for 30 min. If you have 10 µL (1 nanomolar (nM)) use two tubes per hairpin. Check they have been fully evaporated. 

SECTION: JF-669 conjugation to unlabelled hairpins
65. Add 3 µL of 0.1 Molarity (M) Sodium Bicarbonate pH 8-9 to each evaporated hairpin. Mix. 

SECTION: JF-669 conjugation to unlabelled hairpins
66. Add 2 µL of anhydrous DMSO to 0.1 mg of dye. Mix.

SECTION: JF-669 conjugation to unlabelled hairpins
67. Add 2 µL dye mix (100 µg) to each 3 µL of hairpin (10 µg). Mix. It will change colour. 

SECTION: JF-669 conjugation to unlabelled hairpins
68. Leave hairpin-dye mixture to react   at Room temperature in the dark. 

SECTION: JF-669 conjugation to unlabelled hairpins
69. The next morning, add 5 µL nuclease-free water to bring to 10 µL. 

SECTION: JF-669 conjugation to unlabelled hairpins
70. Remove excess dye with a QIAquick Nucleotide removal kit (add 100 µL PN1). 

SECTION: JF-669 conjugation to unlabelled hairpins
71. Elute dye-oligo conjugate in 50 µL nuclease-free water.

SECTION: JF-669 conjugation to unlabelled hairpins
72. Check the hairpin concentration on a spectrophotometer (eg. a NanoDrop One) and dilute to 60 ng/μl.

SECTION: JF-669 conjugation to unlabelled hairpins
73. Separately store hairpins h1-669 and h2-669 in 25 µL aliquot’s in a PCR tube@ -20 °C. 

SECTION: JF-669 conjugation to unlabelled hairpins
74. Test conjugation by HCR.
SECTION: Day 1: Labelling RNA
8.1. Rehydrate with 2 x 5 min wash in 150 µL PBT (0.1%) (1/2).
SECTION: Day 1: Labelling RNA
8.2. Rehydrate with 2 x 5 min wash in 150 µL PBT (0.1%) (2/2).
SECTION: Day 2: Gelation and Proteinase K digestion
17.1. Wash brains 2 x 2 min 150 µL PBT and 1 x 2 min 150 µL PBS.
SECTION: Day 2: Gelation and Proteinase K digestion
17.2. Wash brains 2 x 2 min 150 µL PBT and 1 x 2 min 150 µL PBS.
SECTION: Day 3: Hybridization
30.1. Wash gels 4 x 15 min in 1 mL PBS at Room temperature (1/4).
SECTION: Day 3: Hybridization
30.4. Wash gels 4 x 15 min in 1 mL PBS at Room temperature (4/4).
SECTION: Day 3: Hybridization
30.3. Wash gels 4 x 15 min in 1 mL PBS at Room temperature (3/4).
SECTION: Day 3: Hybridization
30.2. Wash gels 4 x 15 min in 1 mL PBS at Room temperature (2/4).
SECTION: Day 4: Probe Washing
39.1. Wash 3 x 30 min 750 µL Probe Wash Buffer @ 37 °C (1/3).

SECTION: Day 4: Probe Washing
39.3. Wash 3 x 30 min 750 µL Probe Wash Buffer @ 37 °C (3/3).
SECTION: Day 4: Probe Washing
39.2. Wash 3 x 30 min 750 µL Probe Wash Buffer @ 37 °C (2/3).
SECTION: Day 4: Probe Washing
40.1. Wash 3 x 30 min 1 mL PBS @ 37 °C (1/3).

SECTION: Day 4: Probe Washing
40.3. Wash 3 x 30 min 1 mL PBS @ 37 °C (3/3).
SECTION: Day 4: Probe Washing
40.2. Wash 3 x 30 min 1 mL PBS @ 37 °C (2/3).
SECTION: Day 4: Probe Washing
41.1.  Wash 3 x 60 min 1 mL PBS @ 37 °C (1/3).
SECTION: Day 4: Probe Washing
41.3.  Wash 3 x 60 min 1 mL PBS @ 37 °C (3/3).
SECTION: Day 4: Probe Washing
41.2.  Wash 3 x 60 min 1 mL PBS @ 37 °C (2/3).
SECTION: Day 5: Hybridization Chain Reaction (HCR)
47.1. Wash gels 2 x 20 min in 750 µL 5X SSCT @ Room temperature (1/2).
SECTION: Day 5: Hybridization Chain Reaction (HCR)
47.2. Wash gels 2 x 20 min in 750 µL 5X SSCT @ Room temperature (2/2).
SECTION: Day 5: Hybridization Chain Reaction (HCR)
48.1.  Wash gels 2 x 40 min in 1 mL 0.5X SSCT @ Room temperature (1/2).
SECTION: Day 5: Hybridization Chain Reaction (HCR)
48.2.  Wash gels 2 x 40 min in 1 mL 0.5X SSCT @ Room temperature (2/2).
SECTION: Day 6: Mount and Image
50.1. Wash 2 x 30 min with 1 mL PBS-Triton (0.1%) (1/2).
SECTION: Day 6: Mount and Image
50.2. Wash 2 x 30 min with 1 mL PBS-Triton (0.1%) (2/2).
SECTION: Day 6: Mount and Image
51.1. Wash 2 x 30 min with 1 mL PBS (1/2).
SECTION: Day 6: Mount and Image
51.2. Wash 1 x 60 min with 1 mL PBS (2/2).
SECTION: Stripping Probes and Hairpins for Multiplexing
59.1. Wash 4 x 15 min with 1 mL PBS (1/4).
SECTION: Stripping Probes and Hairpins for Multiplexing
59.4. Wash 4 x 15 min with 1 mL PBS (4/4).
SECTION: Stripping Probes and Hairpins for Multiplexing
59.3. Wash 4 x 15 min with 1 mL PBS (3/4).
SECTION: Stripping Probes and Hairpins for Multiplexing
59.2. Wash 4 x 15 min with 1 mL PBS (2/4).
SECTION: Day 6: Mount and Image
54. For gel removal from the holder, incubate gels in 750 µL  of 10% Dextran Sulphate for 20 mins. Gels will shrink and fall off the coverslip.

SECTION: Day 6: Mount and Image
55. For long-term storage, keep gels in 750 µL 10% Dextran Sulphate @ 4 °C