, Incubate the mixture at -80°C for 15 min (not more) or at -20°C for 30 min (do not store probe over-night at this stage)

, Put at -80°C for 15 min (not more) or at -20°C for 30 min (you can stop here and store it at -20°C)

, Paraformaldehyde: Prepare a 20 % (w/v) stock solution in PBS 1X pre-warmed at 65°C

, Use a magnetic stirrer for complete dissolution. Cool on ice and store at -20°C. Warm to 65°C and dilute with PBS to 4% on the day of use

, Keep 4% PFA on ice, use within 24 hours

, Paraformaldehyde 4% with Glutaraldehyde 0.2% in PBT: Mix 80 µl of 25% glutaraldehyde stock solution with 10 ml of paraformaldehyde 4% in PBT

, SSC 20X stock solution: Dissolve 175.3 g NaCl, 88.2 g sodium citrate for 1 L water. Adjust to pH 5 and autoclave

, SDS 20% stock solution: Dissolve 100g electrophoresis-grade sodium dodecyl sulfate for 500 ml water

, tRNA from baker's yeast, 10 mg/ml (Sigma, R-8759), p.20

, Heparin (sodium salt (Grade I-A) from Porcine intestinal mucosa

, Hybridisation Buffer: Solution of formamide 50% (molecular grade or deionised -see 5.3), 5X SSC, 10 µg/ml tRNA, 1% SDS, 50 µg/ml heparin. Aliquot and store at -20°C. Pre-warm for 30 minutes to 65°C on the day of use

, Prepare the day of use, RNase buffer: NaCl 0.5M, Tris-HCl 10 mM pH 7.5, 0.1% Tween 20 in water. 13. RNase A, stock solution. 14. Washing solution 2: 50% formamide, vol.12

, Tween 20 in water. A 10X concentrated solution can be prepared and stored at room temperature once autoclaved

, Prepare a stock solution at 10% in MAB (Maleic acid 0.1M, NaCl 0.15M, pH 7.5) and keep at -20°C 17. Anti-DIG antibody, alkaline phosphatase conjugated, and Fab fragment (Roche) -Wall; to define the wall to limit the semi-automated functions. For example, draw the wall on three planes covering molars or alveolar bone

, Low intensity -air, liquid, and soft tissue

, This will take off the air and soft tissue

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