Cryo-section In Situ Hybridization

Day 1 (DEPC solutions):
Thaw slides
Post fix in fresh 4% PFA/ DEPC PBS 10min. Save the PFA
Wash 2x 5min DEPC PBS
Incubate 2min in 5ug/ml Proteinase K/ DEPC PBS (Time sensitive)
Wash 5min DEPC PBS
Fix in 4% PFA from before 5min
Wash 5min DEPC PBS
Acetylate sections for 10min with acetic anhydride + 0.1M TEA-HCl pH7.5. Add acetic anhydride to TEA immediately before use.
Wash 5min DEPC PBS
Dehydrate 5min in 70% EtOH, 2min in 95% EtOH
Air dry comepletely
Wash 30-45min in Tris/Glycine Buffer
Prepare hybe mix 150-200ul/ slide
Add 1ug/ml probe
Heat hybe with probe to 80-90C, store on ice
Add 150-200ul hybe with probe onto the slide, cover with a hybrislip
Hybe O/N at 65C. Add damp paper towels to prevent drying

Day 2 (No need to DEPC):
Wash 3x 20min 5x SSC RT
Wash 40min 60C 0.5x SSC 20% formamide
Change wash and place at RT until 37C
Wash in NTE 15min 37C
Wash in NTE + 10ug/ml RNase 30min 37C
Wash in NTE 15min 37C
Wash in 0.5x SSC 20% formamide 30min 60C
Wash 2x SSC 30min RT
Place in 1% blocking solution for 10min at least
Add anti-DIG 1:5000 in blocking solution
Incubate O/N at 4C in humidified chamber

Day 3:
Wash slides 4x 10min, 1x 20min in TBS
Wash slides 10min in 0.1% Tween-20, 0.5mg/ml levamisole hydrochloride in water
Warm BM Purple to RT, mix
Add 10ul of 100x levamisole/ tween-20 per 1ml of BM purple
Place about 170ul of staining solution per slide. Cover with hybri-slip. Incubate RT dark.
Stop reaction by washing slides 10min PBS +1mM EDTA
Dry slides mount with DAKO mounting media.

TEA-HCl pH8
Triethanolamine-HCl  3.71g
DEPC H2O  199ml
10N NaOH  900ul
Total  200ml

Acetic anhydride/ TEA-HCL
Acetic Anhydride  125ul
0.1M TEA-HCl pH7.5  50ml

Tris/Glycine buffer
Tris base  24.2g
Glycine  15g
DEPC H2O to 2L

Hybridization mix
40% Formamide
5x SSC
1x Denhard's
100ug/ml fish testis DNA
100ug/ml yeast tRNA

NTE
0.5M NaCl
10mM Tris pH7
5mM EDTA

Blocking
1% Boehringer blocking reagent in MABT

MABT pH7.5
100mM maleic acid
150mM NaCl
0.1% Tween 20

100x Levamisole/ Tween-20
50mg/ml Levamisole
10% Tween-20