Terebratalia in situ for germline genes (+Membranipora testing) 

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First in situ using segmentation genes for Terebratalia transversa. The list is:

Tt netrin BV110 SP6
Tt patched BV115 SP6
Tt hedgehog BV129 SP6
Tt smoothened2 BV131 SP6
Tt nk2.2 BV135 SP6
Tt nk5 BV139 SP6
Mm piwi1 BV94 SP6
Mm gata123

Put in prehybe at 19:45.


Probes for Terebratalia genes were no good, so changing to Vasa, Nanos and Piwi:

Tt vasa
Tt nanos
Tt piwi
Tt vasa (fluo)
Tt nanos (fluo)
Tt piwi (fluo)
Mm piwi1
Mm gata

Put the probes and let hybridizing at hybe temp.


First day of washes. Added Anti-Dig/POD (1:100) to the 3 wells of vasa, nanos, and piwi and the regular Anti-Dig/AP for the remaining; 19h and let overnight at 4° C.


Washing and started developing the regular in situ in 5 wells overnight at 4 °C.


Changes in AP Substrate: 10:30, 12:00, 14:30

AP is becoming purpleish quite rapidly; patterns are developing accordingly fast and as expected for Terebratalia genes. Membranipora Piwi1 shows no sign of staining (although it seems to be a bit pinkish) and GATA123 is appearing, although it is not easy to identify the pattern (or if it is similar to Andi’s in situ).

At 11:00 I started washing and then developing the fluorescent in situ. Checking under the 4D room scope the embryos show staining, but it is hard to say if the in situ worked, yet. I need to look mount them and look in the scope.


Mounted fluorescent in situ samples after staining with DAPI (1:1000) and checked in the scope. Vasa is the strongest, but still a bit weak. So next time it is better to use more probe. Nanos is there, but barely visible and Piwi is difficult to say, since the expression is widespread. Made images in the confocal and the signal seems to be there, but there is a lot of background.

Stopped Vasa regular in situ at 10:00 and changed the substrate for the others. Changed again at 13:30. Yellow crystals were present in the Membranipora wells. Changed at 16:30 and left overnight at 4 °C.


Stopped all wells and stored in 70% glycerol.