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