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).

Positive Control: Gr28bd-G4, TrpA1-G4

Parameters: Light: intensity (500 Lux side, 1000 Lux bottom); frequency = 20Hz; Delay = 1 ms; Duration = 9.9 ms; volts = 6.4

Red lines: completed

mb025b: not selected against tubby

This week we worked on perfecting the fly hooking. Some of the data we got is as follows (Data from just one fly).

The mean ratio of the flies that stay in the middle during the experiments.

Correlation plot between the mean ratio of the flies that stay in the middle versus the Weighted PIs

Slope = 0.0053
Intercept = 0.240
R square value = -0.03834

contrary to the expectations, there seems to be no correlation .

Yellow 1 (Positive Control):  Gr28bd-G4, TrpA1-G4

Parameters:
Light: intensity (500 Lux side, 1000 Lux bottom)

frequency = 20Hz

Delay = 1 ms

Duration = 9.9 ms

volts = 6.4

Yellow 1 (Positive Control):  Gr28bd-G4, TrpA1-G4

Light: intensity (500 Lux side, 1000 Lux bottom), frequency (20Hz)

Lena Matzeder just finished her Bachelor thesis, entitled “Is anxiety a factor influencing photo-preference in Drosophila melanogaster?“. She administered Diazepam (valium) to flies and asked them if that changed their preference for bright or dark places. A warm welcome to Weitian Sun, a Master’s student who will be testing candidate genes for their involvement in operant self-learning.

I have now cleaned the site from all malicious code and installed a few new plug-ins which I still need to properly adjust all settings. The site should now almost be back to normal, please resume posting your data and let me know if there is anything that still needs fixing!

I have just realized that there is no “new comment” section, so we have access only to the last 3 comments. Since there is also no way to have them as rss feeds (only public posts are feeded), it makes it possible to loose track of comments, if somebody (like I just did) look and comment the posts only once in a while.

Is there a way to manage this?

My camera is bigger than Julien’s :mrgreen: I realized that the camera I had been using to track the laser position in my flight simulator didn’t quite fit with the way everything is arranged now. I remembered I had seen an old camera in the basement, so I went there to check it out. It worked really well and it provides a fantastic picture of the fly. It’s just that it is now the largest piece of the equipment: