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Ehler Group

Methods and protocols

Chick heart whole mount preparations (Ehler et al. 1999)

The heart was dissected in cold PBS and fixed in 3% PFA (paraformaldehyde) in PBS with 0.002% Triton X-100 (PBT) for 1 hour.

After several washes in PBT and permeabilisation with 0.1%Triton X-100 in PBS for 30 minutes, the hearts were treated with hyaluronidase (1 mg/ml; Sigma, St Louis, MO, USA) in PBT for 30 minutes at RT (room temperature) in order to remove the cardiac jelly and to ensure access for the antibodies to the inner myocardial wall.

After further washes in PBT and blocking with 5% NGS (normal pre-immune goat serum), 1% BSA (bovine serum albumin) in Gold buffer for 30 minutes, the hearts were incubated with the primary antibody mixtures, diluted in blocking solution, overnight at 4°C.

After 6x 20 min washing in PBT the secondary antibodies were applied for either 6 hours at RT or overnight at 4°C.

The hearts were washed in PBT (6x 20 min) and mounted in 0.1 M Tris-HCl (pH 9.5)-glycerol (3:7) including 50 mg/ml n-propyl gallate as anti-fading reagent.

Same procedure works for mouse embryos, just incubations times have to be increased - enjoy!

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