Nanogold Labeling of the Yeast Endosomal System for Ultrastructural Analyses

The overall goal of the following experiment is to visualize at the ultra structural level the endosomal compartments of the yeast Saccharomyces cei. This is achieved by preparing yeast Sphera plast, which then undergo endocytic uptake of positively charged nano gold particles. As a second step, the sample is fixed and sectioned to obtain ultra thin cryo sections of the cells.

Next, the cryo sections are submitted to the silver enhancement reaction. In order to enlarge and visualize the endocytose positively charged nano gold particles results are obtained that show the morphology of the different yeast endosomal compartments based on the specific labeling of this procedure. The main advantage of this technique of our existing methods, like ImmunoGold labeling of section, is that it doesn't rely on antibodies that often do not work on electromicroscopy preparation.

Although this method can show us insights into the lysosomal system of the yeast sarchi ESIS vei, it can also be applied to other eukaryotic models such as other yeast and mammalian cells. The yeast from which the Sphero Plast will be prepared are first incubated overnight at 30 degrees Celsius in 10 milliliters of the appropriate medium, as determined by the design of the experiment on the following day. After measuring the optical density of the culture at 600 nanometers, dilute the cells in the same culture medium to an OD 600 of 0.2 to 0.4.

Grow the cells to an exponential growth phase with an OD 600 of one to two transfer, 10 D 600 equivalents of cells to a 50 milliliter tube and centrifuge at 2100 Gs for five minutes. After centrifugation, discard the supernatant and resuspend the cell pellet in five milliliters of 100 millimolar pipes and 10 millimolar dihi three at Hall incubate at 30 degrees Celsius for 10 minutes. Collect the cells by centrifugation.

After discarding the supernatant Resus, suspend the cells in five milliliters of medium containing one molar sorbitol, and five milligrams of lytic enzyme. Incubate the mixture at 30 degrees Celsius with gentle shaking for 30 minutes. Centrifuge the cell suspension at 300 Gs for five minutes to collect a pellet fraction which corresponds to the Sphero plats.

Discard the supernatant and re suspend the splats in 960 microliters of ice Cold media containing one molar sorbitol. Transfer the mixture into an ice cold two milliliter micro centrifuge tube. To begin the procedure for nano gold uptake, use a pipette and gently mix the Sphero plast with four of positively charged nano gold particles.

Resus suspended in 40 microliters of water. The final volume of the mixture must be one milliliter. Place the obtained cell suspension on ice for 15 minutes.

Transfer the suspension to room temperature and incubate for the required time to allow nano gold internalization by endocytosis. When the desired nano gold uptake is complete, stop the reaction by adding one milliliter of double strength fixative containing one molar sorbitol to the cell suspension. Keep the tube at room temperature for 30 minutes.

Gently invert the micro centrifuge tube manually several times during the 30 minutes to keep the PHE plast and suspension Next centrifuge twice at 1700 Gs for 25 seconds, discard the supernatant and add one milliliter of fresh standard strength fixative containing one molar sorbitol incubate for two hours at room temperature on a slowly rotating wheel. Subsequently, the cells are processed and cryo sectioned as described in the text protocol prior to starting this procedure, remove the silver enhancement kit from the freezer and thaw in a 37 degrees Celsius incubator. Place the thaw kit in a 24 degree Celsius bench incubator previously placed in the dark room until use.

Place a heating plate in a dark room and warm it to a final temperature of 24 degrees Celsius. Cover the top of the heating plate with a bench coat surface, protector shiny side up and secure it with tape. Place para film on the surface protector and mark the edges with a black marker to enable visualization in the dark.

Tape a thermometer onto the top of the surface protector. To monitor the temperature of the heating plate, gradually adjust the temperature. If it is not a 24 degree Celsius, place a 50 milliliter tube containing double distilled water.

Inside the incubator, fill a small Petri dish with double distilled water, pre-warned at 37 degrees Celsius. Place two nickel grids with the cryo sections in the water specimen side down for 30 minutes. After 30 minutes, fill a second Petri dish with prewarm double distilled water and place the same two nickel grids in the water for another 30 minutes.

Rinse the grids again by passing them specimen side down on several drops of double distilled water at 24 degrees Celsius placed on the parfum that has been fixed on the heating plate. Make sure that nine additional double distilled water drops are ready on the parfum for the rinsing after the silver enhancement reaction. At this point in the procedure, all lights in the dark room must be turned off and the red light turned on.

However, for filming purposes only, the light will remain on. In this demonstration, take out the a and B solutions of the silver enhancement kit from the 24 degrees Celsius incubator in a 1.5 milliliter micro centrifuge tube at six drops of the A solution, followed by six drops of the B solution and mix well with a glass pasti pipette while avoiding bubble formation. Vortex the solution.

Briefly return the a and B solutions to the 24 degree Celsius incubator and take out the C solution. Add six drops of the C solution to the AB mixture. Mix again with a pastier pipette followed by vortexing, and avoid making bubbles.

Use this final mixture immediately. Place drops on the paraform with a pair of clean anti magnetic tweezers. Place the grids on the mixture.

This is the silver enhancing reaction. Leave the grids for six to 15 minutes depending on the enhancement desired for the experiment. Remove the grids from the mixture and pass them rapidly through a succession of six drops of double distilled water placed on Perfil at 24 degrees Celsius for the last three washes.

Leave the grids for seven minutes per drop of water. If performing a real experiment, turn the lights back on when the washing is complete. Subsequently, the results are visualized by either ImmunoGold labeling or membrane staining for an EM investigation.

Different types of yeast endosomal compartments can be labeled with silver enhanced nano gold and visualized by electron microscopy. A five minute uptake of nano gold labels the plasma membrane and early endosomal compartments, such as single vesicles, which are possibly endocytic vesicles and tubular structures, which are very likely early endosomes. A 30 minute incubation with nano gold leads to the labeling of late endosomes or multivesicular bodies.

The resolution power of combining an immuno electron microscopy procedure with this nano gold silver enhancement protocol is underscored by the preservation and quality of the morphology of the shown organelles, in particular, the internal vesicles of the multivesicular bodies. Before using the silver enhancement method, it's necessary to do a pilot experiment to determine the optimal incubation times necessary for both the size and uniformity of these silver particles. Following this procedure.

Other methods like immuno labeling can be performed in order to answer question like localization of particular protein to specific under the mar compartment.