TITLE: Fluorescent in situ hybridization in sponge (Ephydatia muelleri) tissues with tyramide signal amplification
AUTHORS: Vanessa R Ho, April Hill, Sally P Leys

[DESCRIPTION]
Cultured sponges such as Ephydatia muelleri (Demospongiae) have delicate tissues and are attached to coverslips, unlike many other tissue samples such as embryos that are typically free floating. This protocol also uses urea in place of formamide, which is not only safer, but is just as effective at reducing background signals and also improves signal detection. 

It is strongly recommended to have all the reagents prepared at least a day before they are needed. Large quantities of some solutions like PBS can be made at one time but some solutions such as the proteinase K solution are to be prepared fresh.

[STEPS]
SECTION: Tissue fixation and preparation
1. Fix sponges in 4% paraformaldehyde in 1:4 Holtfreter's solution (HS; diluted 1 in 4) overnight
SECTION: Tissue fixation and preparation
2. Wash once in 1:4 HS
 

SECTION: Tissue fixation and preparation
3. Dehydrate the tissues
25% ethanol (EtOH) and 75% 1:4 HS
50% EtOH and 50% 1:4 HS
75% EtOH and 25% 1:4 HS
100% EtOH
SECTION: Tissue fixation and preparation
3.1. Dehydrated tissues can be stored at -80ºC until ready to use.
SECTION: ISH Day 1a - tissue pre-treatment
4. 
 
 Rehydrate sponges - carefully and quickly transfer coverslips into the wells of the cell culture plates
100% EtOH
75% EtOH:25% phosphate buffered saline (PBS)
50% EtOH:50% PBS
25% EtOH: 75% PBS
100% PBS
SECTION: ISH Day 1a - tissue pre-treatment
5. Quench endogenous peroxidases 15 min with 2% H2O2 in EtOH
SECTION: ISH Day 1a - tissue pre-treatment
6. Rinse tissues
PBS x2
2 min PBTw x3
SECTION: ISH Day 1a - tissue pre-treatment
7. 1 min proteinase K digestion
SECTION: ISH Day 1a - tissue pre-treatment
8. Wash with Gly:PBTw solution x2 to stop digestion
SECTION: ISH Day 1a - tissue pre-treatment
9. Acetylation
1% TEA:PBS x2 (Tube #1)
1% TEA:PBS + acetic anhydride (Tube #2) - 3 μL acetic anhydride per 4 mL 1%TEA:PBS 
1% TEA:PBS + acetic anhydride (Tube #3) - 6 μL acetic anhydride per 4 mL 1% TEA:PBS
SECTION: ISH Day 1a - tissue pre-treatment
10. Rinse with PBTw x2
SECTION: ISH Day 1a - tissue pre-treatment
11. Post-fix in 3.7% paraformaldehyde (PFH) + 0.3% gluteraldehyde, at least 1 h at room temperature (RT)
 

SECTION: ISH Day 1b - pre-hybridization
13. Wash with HB 10 min at RT
SECTION: ISH Day 1b - pre-hybridization
14. Replace HB with fresh, pre-heated (55°C) HB, pre-hybridize at 55°C 2-3 h
 

SECTION: ISH Day 1b - probe preparation and hybridization
15. Dilute probe (we use 1:1000; 3 μL 50 ng/μL probe in a final volume of 3 mL HB)
in labeled 1.5 mL Eppendorf tubes, add 3 μL probe to 0.97 mL pre-warmed (55°C) HB
Make sure the lid is shut tight
 

SECTION: ISH Day 1b - probe preparation and hybridization
16. Hybridize 16-72 h at 55°C
 

 

SECTION: ISH Day 2 - post-hybridization stringency washes
17. 
 
Stringency washes - at 55°C
30 min fresh HB
20 min PHB1
20 min PHB2
20 min PHB3
20 min 2X SSC
 

SECTION: ISH Day 2 - post-hybridization stringency washes
18. Block 1 hr blocking buffer (bb) on rocker at RT
SECTION: ISH Day 2 - post-hybridization stringency washes
19. Incubate overnight in antibody (anti-DIG-POD) at 4°C on rocker
Make the stock at 1:500 dilution in bb
Replace half the bb in the well from the previous step with the anti-DIG-POD for final antibody dilution of 1:1000
SECTION: ISH Day 3
20. Wash 20 min MAB x5
SECTION: ISH Day 3
21. Wash 5 min TNT buffer x3
SECTION: ISH Day 3 - signal development - photosensitive steps; keep dark
22. Incubate in 250 μL 1:50 TSA Plus working solution 20 min at RT
 

SECTION: ISH Day 3 - signal development - photosensitive steps; keep dark
23. Stop reaction
Wash 5 min TNT buffer x3 on rocker
Rinse with PBS
SECTION: ISH Day 3 - signal development - photosensitive steps; keep dark
24. Incubate in Hoechst 33342 (1:1000) at 15 min RT for nuclear counterstaining
 

SECTION: ISH Day 3 - signal development - photosensitive steps; keep dark
25. Rinse 5 min PBS X4
 

SECTION: ISH Day 3 - signal development - photosensitive steps; keep dark
26. Mount and store tissue samples
Clear tissues in Mowiol + DABCO anti-fade agent
Mount on microscope slide
Seal with nail polish and let dry
Store at 4°C in the dark
 


 


 


SECTION: ISH Day 1b - pre-hybridization
12. Wash with PBTw x5
SECTION: ISH Day 1b - probe preparation and hybridization
15.1. 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
SECTION: ISH Day 1b - probe preparation and hybridization
15.2. Quickly add probe (final well volume 3 mL)
 

SECTION: ISH Day 2 - post-hybridization stringency washes
17.1. Stringency washes at RT
50% 2X SSC: 50% MAB 10 min
100% MAB x3 (5 min)