Fluorescent in situ hybridization in sponge (Ephydatia muelleri) tissues with tyramide signal amplification

Fix sponges in 4% paraformaldehyde in 1:4 Holtfreter's solution (HS; diluted 1 in 4) overnight

Wash once in 1:4 HS

Dehydrate the tissues

1. 25% ethanol (EtOH) and 75% 1:4 HS
2. 50% EtOH and 50% 1:4 HS
3. 75% EtOH and 25% 1:4 HS
4. 100% EtOH

Dehydrated tissues can be stored at -80ºC until ready to use.

Rehydrate sponges - carefully and quickly transfer coverslips into the wells of the cell culture plates

1. 100% EtOH
2. 75% EtOH:25% phosphate buffered saline (PBS)
3. 50% EtOH:50% PBS
4. 25% EtOH: 75% PBS
5. 100% PBS

Quench endogenous peroxidases 15 min with 2% H₂O₂ in EtOH

Rinse tissues

1. PBS x2
2. 2 min PBTw x3

1 min proteinase K digestion

Wash with Gly:PBTw solution x2 to stop digestion

Acetylation

1. 1% TEA:PBS x2 (Tube #1)
2. 1% TEA:PBS + acetic anhydride (Tube #2) - 3 μL acetic anhydride per 4 mL 1%TEA:PBS
3. 1% TEA:PBS + acetic anhydride (Tube #3) - 6 μL acetic anhydride per 4 mL 1% TEA:PBS

Rinse with PBTw x2

Post-fix in 3.7% paraformaldehyde (PFH) + 0.3% gluteraldehyde, at least 1 h at room temperature (RT)

Wash with PBTw x5

Wash with HB 10 min at RT

Replace HB with fresh, pre-heated (55°C) HB, pre-hybridize at 55°C 2-3 h

Dilute probe (we use 1:1000; 3 μL 50 ng/μL probe in a final volume of 3 mL HB)

1. in labeled 1.5 mL Eppendorf tubes, add 3 μL probe to 0.97 mL pre-warmed (55°C) HB
2. Make sure the lid is shut tight
   Note

   Probe template synthesized using gBlocks Gene Fragments (Integrated DNA Technologies)Reverse primer design includes T7 tail for RNA polymerase bindingPCR amplification of gBlocks using Phusion HF polymerasePCR thermocycler melting temp is 54ºC$$PCR clean up using MinElute column (Qiagen) according to manufacturer's instructionsRiboprobe synthesisMEGAscript T7 transcription kit (Thermo Fisher)DIG-labeling mix (Roche)LiCl₂ precipitation and EtOH extraction

Immediately denature probe after adding to Eppendorf tube by boiling at 80°C for 10 min

• Replace the pre-hyb HB with 2 mL fresh, pre-warmed HB in the wells in the meantime

Quickly add probe (final well volume 3 mL)

Hybridize 16-72 h at 55°C

Stringency washes - at 55°C

1. 30 min fresh HB
2. 20 min PHB1
3. 20 min PHB2
4. 20 min PHB3
5. 20 min 2X SSC
   Note

   Preheat all solutionPHB1 - 75% PHB: 25% 2X SSC pH 7.0PHB2 - 50% PHB: 50% 2X SSC pH 7.0PHB3 - 25% PHB: 75% 2X SSC pH 7.0

Stringency washes at RT

1. 50% 2X SSC: 50% MAB 10 min
2. 100% MAB x3 (5 min)

Block 1 hr blocking buffer (bb) on rocker at RT

Incubate overnight in antibody (anti-DIG-POD) at 4°C on rocker

1. Make the stock at 1:500 dilution in bb
2. Replace half the bb in the well from the previous step with the anti-DIG-POD for final antibody dilution of 1:1000

Wash 20 min MAB x5

Wash 5 min TNT buffer x3

Incubate in 250 μL 1:50 TSA Plus working solution 20 min at RT

Stop reaction

1. Wash 5 min TNT buffer x3 on rocker
2. Rinse with PBS

Incubate in Hoechst 33342 (1:1000) at 15 min RT for nuclear counterstaining

Rinse 5 min PBS X4

Mount and store tissue samples

1. Clear tissues in Mowiol + DABCO anti-fade agent
2. Mount on microscope slide
3. Seal with nail polish and let dry
4. Store at 4°C in the dark