Overlap of FoxP (red) with aPKC or 3 motor neuro lines (D42, C380, OK6) (green)

aPKC

D42

C380

OK6

By comparing the FoxP expression pattern with canidate neurons, VGlut-F-400630 could be an interaction point of FoxP-neurons with the MB.

Cross of FoxPLexA (red) with diffrent GFP-Lines (green)

GMR65A06

GMR20H05

GMR11F02 (dose not overlap)

GMR52B10 not checked yet

aPKC (must be repeated)

MB027B-GAL4/UAS-CD8::GFP (aGFP, aBrp)

MB082C-GAL4/UAS-CD8::GFP (aGFP, aBrp)

MB093C-GAL4/UAS-CD8::GFP (aGFP, aBrp)

MB112C-GAL4/UAS-CD8::GFP (aGFP, aBrp)

MB242A-GAL4/UAS-CD8::GFP (aGFP, aBrp)

MB549C-GAL4/UAS-CD8::GFP (aGFP, aBrp)

MB018B-GAL4/UAS-CD8::GFP (aGFP, aBrp)

MB077B-GAL4/UAS-CD8::GFP (aGFP, aBrp)

MB051B-GAL4/UAS-CD8::GFP (aGFP, aBrp)

MB543B-GAL4/UAS-CD8::GFP (aGFP, aBrp)

MB542B-GAL4/UAS-CD8::GFP (aGFP, aBrp)

MB083C-GAL4/UAS-CD8::GFP (aGFP, aBrp)

MB050B-GAL4/UAS-CD8::GFP (aGFP, aBrp)

MB057B-GAL4/UAS-CD8::GFP (aGFP, aBrp)

MB085C-GAL4/UAS-CD8::GFP (aGFP, aBrp)

MB110C-GAL4/UAS-CD8::GFP (aGFP, aBrp)

MB310C-GAL4/UAS-CD8::GFP (aGFP, aBrp)

MB002B-GAL4/UAS-CD8::GFP (aGFP, aBrp)

MB011B-GAL4/UAS-CD8::GFP (aGFP, aBrp)

MB210B-GAL4/UAS-CD8::GFP (aGFP, aBrp)

MB399B-GAL4/UAS-CD8::GFP (aGFP, aBrp)

MB298B-GAL4/UAS-CD8::GFP (aGFP, aBrp)

MB433B-GAL/UAS-CD8::GFP (aGFP, aBrp)

Fluorescence labeling and microscopy of adult Drosophila mushroom body:
1. Dissection: The brains are removed in cold Phosphate-buffered saline (1X PBS, pH 7.4).
2. Fixation: Tissues are fixed for 20 min in 4% paraformaldehyde (Merck, Germany) in PBST on an orbital shaker at room temperature.
3. Washing: Three quick (each 30 sec) and three long washing steps (each 15 min) and once for 2h with PBST on an orbital shaker at room temperature.
4. Blocking: 5% normal goat serum in PBST are used as a blocking solution for 30 min on an orbital shaker at room temperature.
5. Primary antibody: Brains were incubated for 2 nights in mouse anti-fasciclin II monoclonal antibody (Fas2; 1D4, Developmental Studies Hybridoma Bank, Iowa City, IA) diluted 1:20 in 5% NGS and at 4°C on an orbital shaker.
6. Washing: Brains are washed for 3×30 sec and 4×15 min with PBST at room temperature with orbital shaker.
7. Secondary antibody: Brains are incubated with Goat anti-Mouse IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 594 (Cat#A-11032, ThermoFisher Scientific) at a 1:200 dilution in 5% NGS and PBST for 24hr at room temperature on an orbital shaker in the dark.
8. Washing: Brains are washed for 3×30 sec and 4×15 min with PBST at room temperature with orbital shaker.
9. Mounting: Brains are placed on glass microscope slides and mounted in antifade mounting medium Vectashield® (Vector Laboratories, Burlingame, CA).
10. Slides are stored at 4°C in the dark until microscopy.

Points:
– Use 1X PBS, pH 7.4, 0.3% Triton X-100 (PBST) in step 2.
– Use 10X PBS, pH 7.4, 0.3% Triton X-100 (PBST) in steps 3 to 8.
– Keep the dish containing brains in 1X PBS on ice and fix them within one hour of dissection.
– Transfer the brains using a100 μl pipette.
– Rinse the empty pipette tips twice with PBST (prevent tissues from sticking to the plastic tips).

Control of general FoxP expression patter (line 104y)

Expression of 104y (green) and FoxP (red)

There seems to be no colocalisation of 104y and FoxP.

Overlap of 121y GFP expression with FoxP expression is the fan-shape body.

Problems with line c205, no GFP expression.

I retried the gene-switch method with a new solution of RU486. Since it did not worked before I just tried one line. The flies were left for two days on instant food mixed with a sugar solution of RU486. This time I saw normal GFP expression. So I will test all 4 line in parallel after the break.

I finished my cross for the colocalisation of FoxP with the 6 Gal4-lines i ordered.

Two lines show a nice colocalisation and will be tested.

One would show some overlap, but a completly diffrent expression pattern than it should have. So i will try this line again from the stock to exclude a mixup of the line.

The next one dont seem to have any overlap, but also the pattern looks not like it should.

This line seems to have coexpression but the pattern also looks a bit odd, this may be due to a general weak signal and a bad dissection.

The final line seems to have no overlap with the FoxP expression.