Membranipora in situ with mesodermal genes 

First in situ with mesodermal genes of Membranipora plus some extras. Will add a control with Chema’s probe and do only early stages until late gastrula so I don’t have to worry with cyphonautes larvae.

foxa foxc foxd foxf
mef2 noggin2 eya (long) mprx
fhl mox pax6 wnt4

ProtK lasted 10min30s but washed with 800/500 µL. Otherwise standard protocol. They stayed longer (30min) in the first hybe buffer until the oven was free. Pre-hybe in the oven at 67 °C.

06/04/15

Put probes.

08/04/15

Standard washes as usual. Stayed 1h30 in 1x blocking before I added the antibodies.

09/04/15

Wash off secondary antibody. Started developing. Most genes did not show any clear staining except for pax6 in mid gastrula and foxa. Background was much better than the previous in situ, but not completely absent. Maybe try an even higher temperature?

10/04/15

Now there is visible signal in all fox genes: early single cell expression in early gastrula and in the anterior muscles of the late gastrula. eya expression also came up in some internal cells. Other genes have no clear signal. I stopped all the reactions by the end of the day.

11/04/15

Ethanol washes and placed in 70% glycerol with DAPI.

12/04/15

I’ll mount some slides and check the patterns.