The overall goal of this procedure is to use genetically tagged Centro Somal markers to screen for novel genetic mutations and uncover the function of newly identified Centro somal genes. This can be accomplished by three different imaging strategies. Track a imaging of live testes, track B, chemical fixation of the testes or track C immuno staining.
The first step of the procedure is to isolate the testes of flies expressing genetically tagged centris somal proteins, which can be followed by immediate imaging. The second step is to chemically fix the testes, which can also be followed by imaging. Thirdly, the testes can be immuno stained and then imaged.
Ultimately, the locations of centri proteins are identified by fluorescence microscopy. This method can help answer key questions in the central zone field, such as molecular basis of central formation, central elongation, and central separation. Begin by isolating drosophila testes as detailed by JoVE publication 2 6 4 1.
After isolating the testes, immerse the specimen in six microliters of freshly prepared room temperature dappy staining buffer on a positively charged glass microscope slide. Wait 10 minutes, then gently wash off the excess DPI by replacing the staining buffer twice. With six microliters of PBS.
Be careful to not wash away the testes. Remove the solutions from the slide by using a piece of filter paper to wick it away. Six microliter volumes work well for 18 square millimeter cover slips.
It now helps to use a sharp scalpel to pierce the testes near the cell type of interest, and this minimizes pressure on these cells during the squashing step. Then carefully place a cover slip on top of the specimen and seal it with nail polish. Then mark the position of the testes on the slide with a pen and proceed to imaging.
After isolating the testes, immerse them in a six microliter drop of PBS on top of a positively charged glass microscope slide. Manually orient the testes in a linear fashion or as otherwise preferred. Then use a piece of filter paper to wick away the PBS and replace it with six microliters of freshly prepared fixed buffer.
After five minutes in the fixed buffer, wick it away with a piece of filter paper. Now immerse the specimen in six microliters of freshly prepared DPI staining buffer for 10 minutes. Wash away the DPI by replacing it with two six microliter volumes of PBS.
Then carefully place an 18 square millimeter cover slip on top of the specimen and seal it with nail polish. Then mark the location of the testes on the slide and proceed with imaging. For this procedure first, prepare some siliconized cover slips.
First, immerse the cover slips in a small tray of siliconized solution for a minute at room temperature under a fume hood, make sure that the cover slips are fully exposed and are not stacked on top of each other. Next, wash the cover slips three times in water for one minute per wash. Follow this with three one minute washes in 70%ethanol and a final one minute wash in water.
Then allow the siliconized cover slips to air dry under a fume hood. After dissecting several testes and grave a slide with the specimen type, then add five microliters of PBS to the siliconized cover slip and transfer the testes to the droplet of PBS. Several cover slips each with a single testes are needed to ensure success.
Next, under a dissection microscope, carefully pierce each of the testes as demonstrated earlier. Now, gently place a positively charged glass microscope. Slide over the cover slip, allowing the PBS to become evenly dispersed between the cover slip and the slide.
Then wick away the excess buffer from between the cover slip and slide, which will increase the pressure on the testes and squash them. Now drop the prepared slide into liquid nitrogen. More slides can now be prepared and added to the tank.
Before proceeding using large forceps, remove one of the slides from the liquid nitrogen and quickly use a scalpel to remove the cover slip. The specimen should remain on the slide While removing the cover slip from the specimen. Be careful not to smear it along the surface of the S slide.
This can be achieved by using scalpel to snap the cover slip off in one quick motion. Now incubate the slides in a glass coplan staining jar filled with methanol. Chilled at minus 20 degrees Celsius for 15 minutes.
After the incubation, transfer the slides to a jar containing acetone. Chilled at minus 20 degrees Celsius after 30 seconds in the acetone. Wash the slides for a minute in PBS at room temperature.
Next, incubate the slides for 10 minutes in freshly prepared PBST to block non-specific sites During the PBS TB incubation, fill the wells of a moisture chamber with water After 10 minutes, remove the slides from the PBST and dry each slide around the specimen. Be careful not to dry the specimen itself. As each slide is dried.
Place it in the moisture chamber. It is important to dry the area of the slide surrounding the specimen prior to adding the antibody solution. This ensures that the antibody remains localized on the specimen, thereby preventing drying during the incubation step.
Then gently drop 100 microliters of primary antibody diluted in freshly prepared P-B-S-T-B-R onto the specimens. Use a one to 200 dilution for uncharacterized antibodies. Then place a one centimeter square piece of paraform on top of the specimen to spread the antibody solution and protect the antibody solution from evaporating.
Allow the specimens to incubate for an hour at room temperature. After an hour, use forceps to gently remove the paraform from the slides. Then wash the slides three times in PBST at room temperature for five minutes per wash.
Afterwards, transfer the slides to the moisture chamber with the specimen facing up gently add 100 microliters of secondary antibody diluted in PBST, BR onto the specimens. As before, apply paraform and wait an hour wash off the secondary with three five minute washes in PBST and then three five minute washes in PBS. Then carefully blot the slides dry without touching the specimens.
Add six microliters of mounting media and apply a standard cover slip. After sealing the cover slip with nail polish and marking the position of the testes, proceed with imaging. Centrosomes undergo multiple morphological and functional transformations over the course of spermatogenesis.
One readily observed process is centrosome elongation in spermatogonia, the LAR marker ANA one GFP marks the 0.6 micron long ole this ole elongates during spermatogenesis and reaches a length of 2.5 microns in nearly mature spermatids. Since centri oils of drosophila sperm are uniquely long imaging can be used to make quantitative statements regarding centrosome elongation. Compared to wild type morphology, various mutations that alter central growth have been identified.
Two examples are always early and cannonball mutations that arrest spermatogenesis before the onset of meiosis, but do not block cent elongation in these mutants cent oils of the mature spermatocyte grow to about 2.4 microns in comparison to cent oils of control cells, which reach a maximum length of 1.8 microns. Once mastered track A can be done in 20 minutes, track B in 30 minutes and track C in three to five hours depending on sample size. After watching this video, you should have a good understanding of how to image centrosome in flight.
