Tries with IMARIS
Me and Anders spent some time at the imaris trying to get confidence with the software. We managed to do volume rendering and we counted the FoxP-isoB positive cells
the count resulted really similar to the one I´ve done by eyes:
Protocerebral bridge in FoxP mutated flies
The first 3 images show the protocerebral bridge in FoxP mutated flies. In the second line the first picture shows the protocerebral bridge in a FoxP isoB-Gal4 fly. The last image is taken and modifies from the Virtual Fly Brain
Larvae FoxPB-Gal4 with ELAV or REPO
Larvae ELAV (20x and 40x)
Adults (20x and 40x)
Larvae REPO (20x and 40x)
First try for GABA antibody + FoxP-all_isoforms-Gal4
UNIVERSITY PART
First try for GABA antibody on FoxPisoB-Gal4 adult brain. The reaction is not optimal, there is a lot of background and the signal is too strong. We will have to try again changing conditions in order to be sure of any colocalization.
We also have found some FoxP-all_Isoforms-Gal4 homozygous, we thus wanted to see if there was any macro problem in the overall structure of the brain, but the structure looks fairly normal
Western Blot FoxP-IsoB
KLINIKUM PART
I did the WB again in order to have a better and clearer image, additionally I made a coomassie staining and a silver staining in order to see if there is any contamination in the purified FoxP-IsoB protein
I also made a WB on Drosophila brains using the mouse serum. The signal is not clean but we can see a band of the appropriate size (60 kDa)
WB and New clone for FoxP all isoform
KLINIKUM PART
The serum from the forth injection recognize the FoxP recombinant protein. The band has the appropriate size
UNIVERSITY PART
I have exchanged the Hom1 domain that had a frameshift. Ready to inject again
Titer against FoxP recombinant protein and Immuno
KLINIKUM PART
The sera of the mice (expecially the one immunized with URE-10 peptide) show titer against FoxP recombinant protein. In september we can proceed with the hybridoma using the mouse immunized with ure-10
UNIVERSITY PART
Immunostaining against ELAV and BruchPilot in FoxP-IsoB-Gal4 flies (ELAV: 20x and 40x, BruchPilot 20x)
BruchPilot alone
Cell counting of FoxP-IsoB in stinger staining
Cell counting of 2 adult brains for the Stinger staining in FoxP-IsoB-Gal4 flies. The expression is symmetrical and similar in the 2 brains. I am planning to do some more staining and counting at least 10 brains that I will compare with the FoxP all isoforms-Gal4
