Surface Spreading and Immunostaining of Yeast Chromosomes

The overall goal of this procedure is to visualize chromosome bound proteins by fluorescence microscopy. This is accomplished by first removing the cell wall by treatment with the enzymes. I'm olis.

The second step is to lyce the cell into nuclear membranes by osmotic shock, usually with the help of a detergent. This is done in the presence of a low concentration of fixative to preserve protein structure. After the cell's lye, the concentration of fixative is increased to further stabilize protein structures.

The lysed cells are then deposited on a microscope slide and nuclei allowed to settle onto the slide surface. The final step is to immunostain the samples. The indirect immunos staining method demonstrated here uses a primary antibody that binds the protein of interest and a secondary fluorescently labeled antibody that binds the primary antibody.

Ultimately, immunofluorescence microscopy is used to examine the distribution of chromosome bound proteins. These representative images are of a spread miotic nucleus, immuno stained for the recombination proteins DMC one and RAD 51. The main advantage of this technique over the other commonly used method, which was introduced by Mike Dresser and Craig Deru, is that most of the surface of the slide will contain optimally spread nuclei, making it easy to obtain images of large numbers of spread nuclei from one slide.

This method can help answer key questions in the study of sub nucle structures, such as, do two proteins co localize or does the formation of a particular nuclear substructure depend on the function of a given gene of interest? We emphasize that this method was originally developed by Franz Klein and Yosef Lole. We hope that this video will help others master our version of the technique.

Splats are prepared from yeast cells grown under the desired conditions. Use about two times 10 to the eight cells for each sample. Transfer the cell suspension to 15 milliliter centrifuge tubes.

Spin the tubes in a clinical centrifuge at a setting of five for three minutes to pellet the cells. Decant the medium, taking care not to lose cells from the pellet. Gently resuspend the cells in one milliliter of ZK buffer and then at 40 microliters of one molar dihi.

Three etol incubate for two minutes at room temperature with gentle mixing. Pellet the cells as before resus. Suspend each pellet in one milliliter of ZK buffer.

Add five microliters of a freshly prepared solution of zy liase 100 T that has been thoroughly mixed. Incubate at 30 degrees Celsius for 20 to 30 minutes to remove the cell wall and produce Sphero plats. Asay 10 microliters of the cell suspension under a microscope to determine if sphero plating is complete.

Prior to cell wall removal, yeast cells appear slightly oblong Sphero Platy cells should appear bloated and round. Following the addition of water Sphero plasty cells should lice gently resus bend and wash the cell pellet in 2.5 milliliters of cold MES orbital buffer. After pelleting, the cells again gently resuspend the cell pellet in 300 to 400 microliters of cold MES orbital buffer cells can be kept on ice at this stage for several hours.

This procedure requires a yeast tetra dissection microscope equipped with a 10 x long working distance objective set up in the fume hood, the microdissection needle must be removed. The glass surface upon which chromosomes will be spread should also be prepared ahead of time using a P 20 pipetter pipette 20 microliters of the cell suspension onto the surface of a clean slide with a P 200 pipetter add 40 microliters of a freshly prepared 3%paraform aldehyde 3.4%sucrose solution and gently mix the solution by swirling the slide with the hand until schlein lines disappear. Place the slide under the microscope and confirm that the cells are in focus.

Using a P 200 pipetter, add 80 microliters of 1%Lip Sal swirl as before to mix the solutions as completely as possible and start a timer. If lip sal is not available, the same volume of 1%MP 40 or distilled water may be used. Instead, watch the cells carefully and gently swirl the slide every 15 seconds.

This video clip shows what should be observed when roughly 80 to 90%of the cells have lyed. Stop the timer and immediately remove the slide from the microscope. Lysis should occur from between 30 and 90 seconds.

After starting the timer, add 80 microliters of the paraform aldehyde sucrose solution and swirl to mix. Place the slide on a clean flat surface. Use the side of a clean disposable pasture pipette held below the meniscus of the puddle, but above the surface of the slide itself to spread the liquid across the entire unfrosted surface of the slide.

Do not rake the surface of the slide with the pipette. Use a new pipette for each slide to avoid contamination of samples. Leave the slides to dry overnight in the fume insoluble nuclear components, including the chromosomes will settle onto the slide surface and bind to it once the slides are dry.

Optimal results are obtained by progressing to the immuno staining on the same day. Dip the slides in 0.2%Photo flow for 30 seconds. To remove the dried sucrose solution, lean the slides on an edge with the edge resting on a paper towel to remove residual photo flow.

Dip the slides in one XTBS for five minutes to wash. Remove excess liquid by leaning the slides on an edge, but do not let them dry out. Next, lay the slides frosted side up and pipette 300 microliters of T-B-S-B-S-A onto each slide across the unfrosted portion of the slide.

Incubate the slides in a moist chamber at room temperature for 15 minutes. After 15 minutes, drain the slides by resting a short edge of each slide on a paper towel and leaning the slide against an appropriate support such as a test tube rack. Do not allow the surface to dry immediately.

Apply 80 microliters of T-B-S-B-S-A buffer containing the appropriate dilution of primary antibody for crude rabbit serum. This is typically between a one to 50 and one to 500 dilution. Place a polished 22 by 50 millimeter cover slip on the slide while avoiding bubbles.

Do this by holding the cover slip between the thumb and index finger along the long edge at positions near one short edge. While holding the cover slip at a 30 degree angle relative to the slide. Lower the short edge farthest from the fingers until it rests on the slide just inside the edge of the puddle closest to the frosted region of the slide.

Slowly lower the cover slip in a smooth motion until the fingers contact the slide and then release. Do not attempt to adjust the position of the cover slip once it lands. Place slides in a sealed moist chamber and incubate overnight at four degrees Celsius on the following day, holding the slides by the frosted edge Submerge each in a staining jar containing TBS.

Gently move the submerged slides up and down to remove the cover slip. Try not to use too much force to do this. Wash the slides by submerging in TBS twice for 10 minutes each time.

Replace the buffer after the first wash. Remove the slides from the brack and drain excess liquid by touching their edges to paper towels. Do not let the surface dry immediately.

Add to each slide 80 microliters of T-B-S-B-S-A containing a one to 1000 fold dilution of fluorochrome conjugated secondary antibody. Add the cover slip as before and incubate in the moist chamber at four degrees Celsius for two hours in the dark. After two hours, remove the cover slips wash as before and drain the slides.

Leaning them on paper towels. Allow their surface to air dry for one to two hours in the dark working under subdued light at about 30 microliters of mounting medium containing dbi. And then carefully lower a cover slip.

Wait for two minutes still under subdued light for the cover slip to settle and then seal the edges of the cover slip with clear nail polish. Place the slides in a flat slide box. The slides can be stored in the dark at four degrees Celsius until ready for viewing.

Do not freeze. The slides at this step shown are two examples of optimally spread MYOTIC nuclei double immunostain for the two eukaryotic strand exchange proteins RAD 51 and DMC one and counterstain for DNA with DPI for diploid cells optimally spread miotic nuclei have diameters of approximately five microns in an under spread nucleus. Fewer foci of RAD 51 and DMC one are resolved and not all structures can be viewed in a single focal plane because the sample is not sufficiently flat in overspread nuclei.

Fewer foci of RAD 51 and DMC one remain because of insufficient fixation and or excessive detergent. Treatment experiments were also carried out in which lip sal, which is no longer commercially available with replaced with MP 40 satisfactory results were obtained as shown in the spread miotic nucleus stain for RAD 51 and zip one, a component of the central region of the Synap anal complex. Replacing lip sal with distilled water also yielded satisfactory results.

In both cases, the chromosome compaction that occurs as cells transition from leptin NEA to PAA was largely preserved. This spreading method is suitable for use with super-resolution like microscopy methods. In this image of the Synap Neal Complex, the Paki INE nucleus sustained for zip one and red one.

Components of the Synap Neal Complex and viewed by stimulated emission depletion microscopy. Note that the zip one antibody used here shown in green binds near the lateral elements as evidenced by the parallel linear staining pattern. The lateral elements are about 100 nanometers apart and cannot be resolved by conventional microscopy.

Once mastered, the sphere of plating and spreading technique can be done in two to four hours depending on the number of samples with practice, up to 100 slides can be prepared by this method. It is advisable to prepare one or more duplicate slides from each sample. The duplicates can be stored for future use at minus 20 degrees Celsius after the overnight drying step.

The slides usually keep for years when stored. This Way.