JOURNAL OF ELECTRON MICROSCOPY TECHNIQUE 17:471-472 (1991)
Rapid Communication A Plexiglass Perfusion Chamber for Staining Multiple Electron Microscope Grids Zhongrong Luo and John M. Robinson Department of Cell Biology, Neurobiology, and Anatomy Ohio State University Columbus, OH 43210 We h a v e developed a sealed-perfusion chamber for the incubaticn of multiple electron microscope grids. This chamber has been used for (1) incubation of whole mount cells (cultured on Formvar-coated grids) for enzyme cytochemical reactions, and for (2) heavy metal staining of thin sections. The chamber has several advantages for each application. The flow-through plexiglass chamber is shown in Figures 1,2. The chamber was constructed of a thicker base-section (80 x 80 x 18 mm) in which a square depression was machined (44.5 x 44.5 x 2 mm) for holding the grid supporting plate. Within that, another depression was machined (30 x 30 x 6 mm) which serves as the fluid reservoir (= 7 ml) for staining grids. A g r o o v e was machined f o r housing a gasket (neoprene, 0.25 mm) which prevents leakage. Aligned entrance and exit ports (4 mm diam.) were machined into the base plate. The top section was made of a thinner piece of plexiglass (80 x 80 x 5 mm). The base has screw threads (1224 inch scale) machined into each corner. The top and base sections can be secured by tightening brass thumb screws. Grids are placed into thin slots which were cut into a supporting plate made of a flat square piece of clear flexible plastic as described by Hiraoka (1972) (purchased from Polysciences, Warrington, PA). A vise-like device, similar to that of Schultz (1987), was constructed which allows for the controlled deformation of the flexible plastic (see Fig. 3). The grids are securely held in place after the vise is released, as the flexible plastic returns to its original flat conformation. The flexible plastic can then be inverted into the reservoir space so that the grids a r e oriented parallel to the direction of the flow of the perfusate. T h e chamber proved to be an excellent device for carrying out enzyme cytochemical reactions on whole cells cultured on Formvarcoated EM grids. In this case all of the incubation steps and washes can be conducted with the chamber in a water bath for maintenance of optimal temperature. In addition, agitation of the cytochemical medium can be achieved by the gentle back and forth movement of the plunger of C1991 WILEY-LISS, INC.
a syringe attached to the chamber by means of tubing. Changes of solution are readily accomplished by withdrawal of a solution into a syringe and replacement by injection of a solution from another syringe or alternatively by the perfusion of solutions through the chamber. An example of a whole mount cytochemical reaction carried out in this device is shown in Figure 4 (see figure legend for details). The only problem which we have encountered using this system for enzyme cytochemical reactions is that care must be taken to prevent cells from drying during the time they are being loaded into the grid supporting plate. To prevent this, we put the vise into ice and place small drops of buffer on the cell side of the Formvar-coated grids while loading them into the plate. The chamber is equally well suited for routine staining of thin sections with heavy metals (i.e., uranyl acetate and lead citrate). For such staining the chamber is held in a vertical position by a clamp which is, in turn, attached to a stand. This position facilitates removal of all visible air bubbles during filling and permits rinsing of the tubing with water from a bottle mounted above the chamber. This chamber has the additional advantage of being air tight and thus minimizes the precipitation of lead stains due to atmospheric carbon dioxide. It should be noted that a relatively small volume of stain is used and that it can be returned to the delivery syringe for safe containment. Changing solutions for staining and washing is a very g e n t l e process such that even Formvar films on single slot EM grids remain intact during this staining procedure. Up to fifty grids at a time can b e used in this chamber. Furthermore all of these grids will be stained under exactly the same conditions; this may be important for comparative purposes. It is, of course, possible to construct larger or smaller chambers as the need arises.
References Hiraoka, J. (1972) A holder for mass treatment of grids, adapted especially to electron staining and autoradiography. Stain Tech., 47:297-301. Schultz, R.L. (1987) Mini-vise aids section staining. EMSA Bulletin 17:91-92. Received November 16, 1990; accepted in revised form December 6, 1990
472
Z. LUO AND J.M. ROBINSON
Figure 1. The component parts of the disassembled staining chamber. Figure 2. The vise used for bending the flexible plastic. This controlled bending greatly facilitates loading of grids -Figure 3. The staining chamber mounted on a stand with the perfusate being delivered from below via a syringe. A wash bottle containing distilled water is mounted above the chamber. A beaker for collecting the washing solution is also shown.. Figure 4. Whole mount EM of cultured resident murine macrophage which was stimulated by phorbo1-12-myristate-13-acetateand then incubated in the perfusion chamber for the demonstration of acid phosphatase activity . Note that under these conditions the reaction product is present in thin tubular structures (arrows). Bar is 2 pm. Supported by Grants from the NIH, Council for Tobacco Research, and American Heart Association.
Rapid Communication A Plexiglass Perfusion Chamber for Staining Multiple Electron Microscope Grids Zhongrong Luo and John M. Robinson Department of Cell Biology, Neurobiology, and Anatomy Ohio State University Columbus, OH 43210 We h a v e developed a sealed-perfusion chamber for the incubaticn of multiple electron microscope grids. This chamber has been used for (1) incubation of whole mount cells (cultured on Formvar-coated grids) for enzyme cytochemical reactions, and for (2) heavy metal staining of thin sections. The chamber has several advantages for each application. The flow-through plexiglass chamber is shown in Figures 1,2. The chamber was constructed of a thicker base-section (80 x 80 x 18 mm) in which a square depression was machined (44.5 x 44.5 x 2 mm) for holding the grid supporting plate. Within that, another depression was machined (30 x 30 x 6 mm) which serves as the fluid reservoir (= 7 ml) for staining grids. A g r o o v e was machined f o r housing a gasket (neoprene, 0.25 mm) which prevents leakage. Aligned entrance and exit ports (4 mm diam.) were machined into the base plate. The top section was made of a thinner piece of plexiglass (80 x 80 x 5 mm). The base has screw threads (1224 inch scale) machined into each corner. The top and base sections can be secured by tightening brass thumb screws. Grids are placed into thin slots which were cut into a supporting plate made of a flat square piece of clear flexible plastic as described by Hiraoka (1972) (purchased from Polysciences, Warrington, PA). A vise-like device, similar to that of Schultz (1987), was constructed which allows for the controlled deformation of the flexible plastic (see Fig. 3). The grids are securely held in place after the vise is released, as the flexible plastic returns to its original flat conformation. The flexible plastic can then be inverted into the reservoir space so that the grids a r e oriented parallel to the direction of the flow of the perfusate. T h e chamber proved to be an excellent device for carrying out enzyme cytochemical reactions on whole cells cultured on Formvarcoated EM grids. In this case all of the incubation steps and washes can be conducted with the chamber in a water bath for maintenance of optimal temperature. In addition, agitation of the cytochemical medium can be achieved by the gentle back and forth movement of the plunger of C1991 WILEY-LISS, INC.
a syringe attached to the chamber by means of tubing. Changes of solution are readily accomplished by withdrawal of a solution into a syringe and replacement by injection of a solution from another syringe or alternatively by the perfusion of solutions through the chamber. An example of a whole mount cytochemical reaction carried out in this device is shown in Figure 4 (see figure legend for details). The only problem which we have encountered using this system for enzyme cytochemical reactions is that care must be taken to prevent cells from drying during the time they are being loaded into the grid supporting plate. To prevent this, we put the vise into ice and place small drops of buffer on the cell side of the Formvar-coated grids while loading them into the plate. The chamber is equally well suited for routine staining of thin sections with heavy metals (i.e., uranyl acetate and lead citrate). For such staining the chamber is held in a vertical position by a clamp which is, in turn, attached to a stand. This position facilitates removal of all visible air bubbles during filling and permits rinsing of the tubing with water from a bottle mounted above the chamber. This chamber has the additional advantage of being air tight and thus minimizes the precipitation of lead stains due to atmospheric carbon dioxide. It should be noted that a relatively small volume of stain is used and that it can be returned to the delivery syringe for safe containment. Changing solutions for staining and washing is a very g e n t l e process such that even Formvar films on single slot EM grids remain intact during this staining procedure. Up to fifty grids at a time can b e used in this chamber. Furthermore all of these grids will be stained under exactly the same conditions; this may be important for comparative purposes. It is, of course, possible to construct larger or smaller chambers as the need arises.
References Hiraoka, J. (1972) A holder for mass treatment of grids, adapted especially to electron staining and autoradiography. Stain Tech., 47:297-301. Schultz, R.L. (1987) Mini-vise aids section staining. EMSA Bulletin 17:91-92. Received November 16, 1990; accepted in revised form December 6, 1990
472
Z. LUO AND J.M. ROBINSON
Figure 1. The component parts of the disassembled staining chamber. Figure 2. The vise used for bending the flexible plastic. This controlled bending greatly facilitates loading of grids -Figure 3. The staining chamber mounted on a stand with the perfusate being delivered from below via a syringe. A wash bottle containing distilled water is mounted above the chamber. A beaker for collecting the washing solution is also shown.. Figure 4. Whole mount EM of cultured resident murine macrophage which was stimulated by phorbo1-12-myristate-13-acetateand then incubated in the perfusion chamber for the demonstration of acid phosphatase activity . Note that under these conditions the reaction product is present in thin tubular structures (arrows). Bar is 2 pm. Supported by Grants from the NIH, Council for Tobacco Research, and American Heart Association.
