Whole mount ISH on axolotl tissue

1. fix embryo or tail blastema in MEMFA (0.1M MOPS pH7.4, 2mM EGTA, 1mM MgSO4, 3.7% formaldehyde) 2h to over night at room temperature
2. store at –20C in 100% MeOH or EtOH (prevents lipid droplet in blastocoel to stain dark purple)
3. rehydrate tissue by 2-5min washes in MeOH,

<margin-left: 50px> * 75%MeOH 25% H2O

• 50%MeOH 50%H2O
• 25%MeOH 75%PTw (PBS + 0.1%tween20)
• 100% Ptw

1. treat tissue with ProteinaseK (10µg/ml 10-15min RT for embrys and tail blastema, 30µg/ml 30min at 37C for limb blastema)
2. rinse twice in 0.1M triethanolamine pH7.8 then add 0.5% acetic anhydrate for 10min (can add 12.5µl acetic anhydrate to 5ml triethanolamine for 5min and then add another 12.5µl for additional 5min)
3. rinse twice with Ptw
4. refix in 4% foramaldehyde in Ptw for 20min and rinse in Ptw
5. add 50% hybridization buffer 50% Ptw, replace with 100% hybridization buffer (50% formamide, 5x SSC, 1mg/ml yeast RNA, 100µg/ml heparin, 1x Denhardt’s, 0.1% tween20, 0.1% CHAPS, 5mM EDTA)
6. pre-hybridize at 60-65C over night (at least 6h! Xenopus embryo 60C, Hoxc10 axolotl tail blastema 65C)

1. heat the Dig-labeled probe (10µg/ml in hyb) to 95C for 30min, dilute to 1µg/ml in hyb and add to sample for hybridization at 60-65C 24-72h

1. remove probe and keep (can be recycled 2 times)
2. wash with hyb 60-70C 10min (axolotl tail 70C)
3. wash 3x in 2xSSC 60-70C, 20min each wash
4. optional: wash in 2xSSC with RNaseA 20µg/ml, RNaseT1 10U/ml 37C 30min (RNaseA dissolve 10mg/ml in TE, boil 10min, RNaseT1 10.000U/ml boiled in 0.1M Na acetate pH5.5, store in frozen aliquots!) remove RNase by washing once in 2xSSC at RT
5. wash twice in 0.2xSSC 30min 60-70C
6. wash twice in Maleic acid buffer (MAB): 100mM maleic acid, 150mM NaCl pH7.5. wash 10-15min at room temperature
7. replace with MAB + 2% Boehringer Mannheim Blocking Reagent (dissolves by heating, but always cloudy). Wash 15min
8. wash another time for1h at RT (preincubation, optional with 20% heat inactivated lamb or sheet serum at 4C over night)
9. replace with fresh solution of MAB+2%BMB containing a 1/2000 dilution of sheep anti dig antibody coupled to alkaline phosphatase, rock over night at 4C or 4h at RT (shorter is better)
10. to remove excess ab wash embryos at least 5 times, 1h each at RT with MAB, one wash can be done over night
11. for chromogenic reaction was twice, 5min each at RT, with alkaline phosphatase buffer (100mM TRIS pH9.5, 50mM MgCl2, 100mM NaCl, 0.1% tween20, 2mM levamisol add freshly before use!!!)
12. replace with BM purple supplemented with levamisol or with AP buffer containing 4.5µl NBT (75mg/ml in 70% dimethyl formamid) and 3.5µl BCIP (50mg/ml in 100% dimethyl formamid) per ml of buffer
13. colour reaction is visible form 5min to 3days, can look at tissue in AP buffer, when reaction finished fix in MEMFA, can refix over night
14. rinse in 70% EtOH, store in MeOH at –20C
15. tissues can be cleared in methyl salicylate for photography
16. for sectioning whole mount tissues rehydrate in PBS, freeze in OCT and cryosection at 40µm thickness