Regular and fluorescent in situ for Terebratalia segmentation genes


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Beginning a regular in situ for the newest wnt7 and wnt11 and for en and dlx to get their patterns. Also testing the DNP-POD for en and DIG-POD for dlx, wnt1, and wnt5. One well will be for a first double in situ with en and wnt5.

DNP-AP >> en dlx wnt7 wnt11
DIG-POD >> en (DNP-POD) dlx wnt1 wnt5
DOUBLE en (DNP-POD) + wnt5 (DIG-POD)

16/02/13

All probes diluted to a regular concentration of 1 ng/µL and added at 14h30.

18/02/13

Maybe the oven was not rocking? Not sure…

Did all the washes and added the corresponding antibodies at the following concentrations:

Anti-Dig-AP 1:5000
Anti-Dig-POD 1:200
Anti-DNP-POD 1:500

19/02/13

Just washes.

20/02/13

Dig-AP started to develop at 12h00. engrailed and distal-less were already well defined in 30 minutes while wnt7 had nothing and wnt11 had a faint band in pedicle lobe. The patterns became strong after a few hours. AP substrate exchanged once and left overnight at 4°C.

For fluorescent samples… chaos begin. I mistakenly made the TNT buffer with 1% Tween, instead of 0.1%. After developing I split the samples for en, dlx, wnt1, wnt5 into Ptw and detergent treatments. Reactions were stopped with 2x of detergent solution for 2 minutes at 60°C. I also washed the samples with the 1st POD inactivation inadvertently. At the end I see none of the expected expression patterns under the fluorescent lamp. DNP samples seem to have their mesoderm stained while Dig-POD are fluorescent in the gland cells of the epidermis.

Will incubate the second probe overnight, anyway.

21/02/13

Realized I used a regular Cy3 antibody (with Anti-DNP-HRP rabbit) and not the TSA Kit Cy3… for sure this was the issue. I developed the second probe with Cy5, but now I cannot check if it worked, because the red filter is not installed in the scope.

Ttra_mesoderm

After developing the second probe for the doubles, the in situ worked! So engrailed-DNP does work well even for the second probe. Pictures taken under the fluorescent scope:

en_early_larva en_larva