Double in situ in Terebratalia


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Starting double in situ with probes correctly diluted to 1 ng/µL. Stages sampled are: late blastula, early/mid/late gastrula, trilobed, early/late larva.

en-DNP+wnt1-DIG-POD en-DNP+wnt5-DIG-POD

Regular beginning. I left 5 minutes of the ProtK shaking, mistakenly.

11/03/13

Regular washing. Put Anti-DIG-POD diluted 1:250 (in 250 µL each well).

12/03/13

Began washing at 11h30. Did extra wash with PBT and also a couple of extras for PTw.

13/03/13

Developed first probe with Cy3 and stopped the reaction with 1 detergent wash of 5 min and another detergent wash of 10 min. Specific expression can be seen in the stereoscope. Put the Anti-DNP antibody for the second probe.

14/03/13

Just washed thoroughly.

15/03/13

Develop the second.

WORKS! engrailed=green, wnt1=red

en+wnt1