Lineus ruber and Membranipora in situ

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Starting the first in situ in the nemertean Lineus ruber and trying different genes in Membranipora after establishing the ProtK timing. Selected genes are:

L. ruber foxa six3/6 cdx gsc
M. membranacea bra gsc piwi1 vasa

All wells treated with 10 min in 1x protk concentration (0,01 mg/mL).


Added probes.

19 and 20/11/12

Washing days, normal activity. Samples stayed in blocking buffer for 1h30 (instead of 1h) due to epic foosball match.


Began developing at 11h. Twenty minutes later Mm bra cyphonautes shell were already completely blue… It seems that mucus cells in Lineus juveniles are being stained. No real signal coming up, except in six3/6, maybe. Mm piwi1 and vasa look like to be working, something is appearing. Exchanged AP once and let it at 4°C.


Changed the AP of Lineus twice. Some signal kind of visible below the epidermis, nothing in the embryos. Stopped Membranipora development.


Patterns in Lineus more visible and apparently real signal. foxa expressed in the gut region, six3/6 in the brain/anterior, cdx in the posterior tip (not in the epidermis), and gsc in the anterior mouth region. Let developing one more day at 4°C.


Stopped reaction. Background began to come up, but I could check the expression more clearly. For example, foxa is detected in the mouth region and cdx in the anus. Now what needs to be done is remove the mucus cells with cystein before fixation.


Restart development of Lineus to see if the signal get stronger in the juveniles and something appears in the embryos.


Finally stopped the development. Will do ethanol washes tomorrow.