View source for Whole mount ISH on axolotl tissue

# 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
# store at –20C in 100% MeOH or EtOH (prevents lipid droplet in blastocoel to stain dark purple)
# rehydrate tissue by 2-5min washes in MeOH, 
* 75%MeOH 25% H2O
* 50%MeOH 50%H2O
* 25%MeOH 75%PTw (PBS + 0.1%tween20) 
100% Ptw
# treat tissue with ProteinaseK (10µg/ml 10-15min RT for embrys and tail blastema, 30µg/ml 30min at 37C for limb blastema) 
# 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)
# rinse twice with Ptw
# refix in 4% foramaldehyde in Ptw for 20min and rinse in Ptw
# 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 
# pre-hybridize at 60-65C over night  (at least 6h! Xenopus embryo 60C, Hoxc10 axolotl tail blastema 65C)

# 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

# remove probe and keep (can be recycled 2 times)
# wash with hyb 60-70C 10min (axolotl tail 70C)
# wash 3x in 2xSSC 60-70C, 20min each wash
# 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
# wash twice in 0.2xSSC 30min 60-70C
# wash twice in Maleic acid buffer (MAB): 100mM maleic acid, 150mM NaCl pH7.5. wash 10-15min at room temperature
# replace with MAB + 2% Boehringer Mannheim Blocking Reagent (dissolves by heating, but always cloudy). Wash 15min
# wash another time for1h at RT (preincubation, optional with 20% heat inactivated lamb or sheet serum at 4C over night)
# 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)
# to remove excess ab wash embryos at least 5 times, 1h each at RT with MAB, one wash can be done over night
# 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!!!)
# 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
# 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
# rinse in 70% EtOH, store in MeOH at –20C
# tissues can be cleared in methyl salicylate for photography
# for sectioning whole mount tissues rehydrate in PBS, freeze in OCT and cryosection at 40µm thickness