Mounting Adult Drosophila Brains: A Method to Prepare Slides for Confocal Imaging

- On a positively-charged microscope slide, adhere two cover slips with enough distance between them to create a space for the Drosophila brains to be mounted. Under the stereo microscope, pipette the brains in mounting medium into that space. The negative charge of the tissue will help the brains adhere to the positive charge on the surface of the slide.

Remove excess mounting medium to allow for precise positioning of the brains on the slide in a grid pattern with the antennal lobes facing up, then bridge the two cover slips by adhering a third cover slip across to cover the brains.

To fill in the cavity, place mounting medium one drop at a time into the edge of the cavity, and allow it to spread by capillary motion so that it does not disturb the brains.

Seal the cavity with nail polish, and store at negative 20 degrees Celsius in the dark to preserve the fluorescence of stained tissues.

In the example protocol, we will mount Drosophila brains for confocal imaging of immunostained neurons.

- To mount the brains, build a bridge slide. Position two base covers slips roughly one centimeter apart on a positively-charged slide. Make certain that the positively-charged side is face up. Then adhere the cover slips to the slide with fingernail polish, and let the polish dry completely before proceeding.

Next, place the slide under a stereo microscope and pipette brains in medium into the space between the cover slips. Be sure to adjust the lighting to improve visualization of the brains.

Next, aspirate the extra mounting media from the slide, being careful to avoid the brains. Then, wick away remaining excess mounting media. This will allow the brains to be positioned more precisely. Now, using forceps, orient the brains into a grid pattern with their antennal lobes facing up.

Then, place a cover slip over the brains, and use fingernail polish to seal the edges of the top cover slip that are attached to the base cover slips.

Now, load the center cavity with fresh mounting media drop-wise, allowing the media to be drawn under the cover slip by capillary action. When the cavity is filled, seal it in completely using clear fingernail polish.