16 Whole Mount in Situ Hybridization for mRNA Detection in Chick Embryos
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Posthybridization washing and antibody reaction
Note: All washes are carried out with sufficient shaking to agitate
the embryos, otherwise high backgrounds result.
We usually use Iml solution in a well of the 24-well dish.
After each 65°C wash, put the 24-well dish on a heater block at
65°C, then change supernatant individually so samples don't
cool. Keep solutions I and III at 65°C in hybridization oven.
1. Wash with solution I, three times for 20 mins at 65°C.
Note: Diluted probe can be stored at -20°C and reused.
2. Wash with solution III, twice for 5 mins at 65°C.
3. Wash with solution III, three times for 20 mins at 65°C.
4. Rinse with TBST, twice for 10 mins at RT.
5. Preblock the embryos with 1.5% blocking reagent in TBST for
60-90 mins.
6. During this, preabsorb the antibody for Ihr with embryo powder as described below.
1) Weigh out 3mg embryo powder into a microtube, add 0.5ml
TBST and heat at 70°C for 30 mins.
2) Coolon ice and add 5)111.5% blocking reagent and 1)11 APanti-DIG-antibody.
3) Shake gently at 4°C for Ihr, then spin in a microfuge at 4°C
for 10 mins. Recover the supernatant and dilute it to 2 ml with
1.5% blocking reagent.
7. Incubate with the diluted AP-anti-DIG antibody and rock
overnight at 4°C.
Post antibody reaction wash and color reaction
1. Wash with TBST, four times for 5 mins.
Note: Use TBST containing 1 % Tween-20 in this step. The higher
concentration of Tween-20 helps to give low backgrounds.
2. Wash with TBST, five to seven times for Ihr.