In situ Mm Nanos and Tt Vasa, Nanos, Piwib

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Trying 2 samples of late cyphonautes of Membranipora, one without proteinase-k treatment and one with 30s half-concentrated dose. Also, samples of all stages of Terebratalia using the probes for Vasa, Nanos, and Piwib.

Mm Nanos Mm Nanos Tt Vasa Tt Nanos Tt Piwib
no prot-k 30s half-conc prot-k 10min prot-k 10min prot-k 10min prot-k

Membranipora embryos/larvae take 1 min to sink. When trying to do 30s of prot-k I managed to start removing the solution at 15s, but only finished and added glycine around 45s. I added a double dose of glycine to dilute the prot-k well and quickly did the second wash (also with double amount). Membranipora embryos treated with prot-k were fixed for 1h30 while Terebratalia for 1h.


Added the probes to the wells and let it hybridize over the weekend.


First day of washes. Membranipora embryos look ok (not dissolving) and Terebratalia became a bit more transparent, but also look integer. Put the antibody at 18:30, incubating overnight rocking at 4 °C.


Washes with PBT and PTw ran as expected. Letting overnight at 4 °C. Embryos are still there and apparently no differences between prot-k treatment and control of Membranipora.


Developing from 11h.

30min: Apparently nothing on Membranipora and Terebratalia Nanos and Piwib. Terebratalia Vasa is already showing the expression, a bit more broadly expressed in gastrula stages and restricted to a ring in the mid-lobe of larvae.
60min: Nothing on Mm. Tt Nanos and Piwib started to develop a faint staining in the archenteron region of the gastrula. Tt Vasa continues to show a strong staining pattern.
90min: Same.
3h: TtVasa looks stronger and TtNanos starting to appear more clearly.
5h: Same pattern. Changing the AP substrate and leaving at 4 °C overnight.


Stopped Tt Vasa at 10h (following PTw washes). Changed the AP substrate for the others. Signal is stronger in Nanos and Piwib, better revealing the pattern. Apparently nothing is coming up in Mm, although I saw some unspecific bilateral staining appearing.

Changed AP substrate at 10h, 14h, and 18h. Leaving overnight at 4°C.


Forgot to filter the AP substrate, so wells were full of yellow crystals in the bottom and covering the samples… :( I stopped the reaction and did the Ethanol washes for all wells. Crystals were mostly dissolved, but wells became dirty. After ethanol washes it was all good again. Stored in the fridge with 70% glycerol in PTw.


Mounted Membranipora samples with and without proteinase-k. Both look similar and show no expression of Nanos in the expected region. So it seems that the in situ did not work again. At least the embryos made it to the end of the protocol in one piece.