Mounting Adult Drosophila Brains: A Method to Prepare Slides for Confocal Imaging
Transcrição
– On a positively-charged microscope slide, adhere two cover slips with enough distance between them para create a space for the Drosophila brains para be mounted. Under the stereo microscope, pipette the brains de mounting medium into that space. The negative charge of the tissue will help the brains adhere para the positive charge on the surface of the slide.
Remove excess mounting medium para allow for precise positioning of the brains on the slide de a grid pattern with the antennal lobes facing up, then bridge the two cover slips by adhering a third cover slip across para cover the brains.
To fill de the cavity, place mounting medium one drop at a time into the edge of the cavity, and allow it para 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 de the dark para 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. Posição 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 para the slide with fingernail polish, and let the polish dry completely before proceeding.
Next, place the slide under a stereo microscope and pipette brains de medium into the space between the cover slips. Be sure para adjust the lighting para improve visualization of the brains.
Next, aspirate the extra mounting media from the slide, being careful para avoid the brains. Then, wick away remaining excess mounting media. This will allow the brains para 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 para seal the edges of the top cover slip that are attached para the base cover slips.
Now, load the center cavity with fresh mounting media drop-wise, allowing the media para be drawn under the cover slip by capillary action. When the cavity is filled, seal it de completely using clear fingernail polish.
