In situ segmentation genes Terebratalia

Starting an in situ for segmentation genes in Terebratalia and repeating the fluorescent in situ for Vasa and Nanos, trying to reduce the background. The plate looks like this:

netrin patched hedgehog vasa
smo2 nk2.2 nk5 nanos

For vasa and nanos I will use a lower concentration of the antibody, 1:500 (last time it was 1:100) — effort to reduce background. For nanos I will also double the amount of probe to get a stronger signal.

Samples put in prehybe at 17h:00.


Added regular probes at 1 ng/µL, except for nanos which was 2 ng/µL (trying to increase the signal/noise ratio for fluorescent in situ).


Washes. Added regular Anti-Dig/AP to segmentation genes (1:5000) and Anti-Dig/POD (1:250) to vasa and nanos (in an attempt to lower the background noise — used 1:100 previously).


Regular washes with PBT and PTw.


Started developing segmentation genes of Terebratalia at 11:30. For the fluorescent in situ I developed normally for 1h30 and then stopped the reaction with a detergent solution used by (10.1038/nature10838) to reduce the background; 3 washes of 20 min at 62 °C. The solution:

[list of chemicals]

I checked the slides in the fluorescent lamp and did not see any staining. It is also not so clear if there is less background. I guess I need to check in the confocal.

The segmentation genes began to appear. Netrin is the strongest one and the pattern is interesting. I notice that the “ring” background might be covering the whole anterior of the pedicle lobe. Other genes are still weak to say something.

Changed the AP substrate at 16:30 and let it developing at 4 °C.


Patterns are more clear today. Changed the substrate 3 times (10:15, 12:30, 16:00) and let it overnight at 4 °C. Crystal began to appear after lunch and are already covering the whole bottom of the well.

01/06 – 04/06

Stopped netrin, nk2.2, and nk5. Developed others until 04/06. Many crystals forming…