Light microscopy antibodies

Labeling antibodies with colloidal gold was developed for electron microscopy, but the procedure can be used for light microscopy as well. 

Opening this section, we wrote that immunohistochemistry at the ultrastructural level is based on the same principles as immunohistochemistry for light microscopy. The same is also true for the specificity controls. Obligatory controls are (1) omission of incubation with primary antibodies, (2) substitution of primary antibodies by the corresponding IgG at the same final concentration, and (3) incubation in media containing primary antibodies that have been preincubated at room temperature for 2 h with a ten-fold molar excess of the corresponding control... 

Figure 11.2 Morphological differences between human alveolar epithelial cells in primary culture (A and C) and the A549 cell line (B and D). Cells are visualised by light microscopy (A and B) and immunofluorescence microscopy (C and D) using an antibody against a tight junctional protein, occludin.

The localization of proteins and carbohydrates within cells and tissues with specific antibodies has long been proven to be a valuable technique. Immuno-localization procedures allow one to detect not only well-characterized cellular structures but also provide information about newly characterized proteins and carbohydrates. This chapter will review some of the advantages and drawbacks of common chemical fixation and permeabilization methods used for immuno-localization at the level of light microscopy. 

Fritz, P, Hoenes, J, Schenk, J, Mischelinski, A, Grau, A., Saal, J G., Tuczek, H. V, Multhaupt, H., and Pfleiderer, G (1986) Color development of unmunogold-labelled antibodies for light microscopy. Histochemistry 85, 209-214. 

All incubations in antibody/avidin/enzyme conjugates described below are carried out at room temperature for 30 min unless otherwise stated. The substrate reactions are carried out at room temperature, and signal development monitored by light microscopy. The substrate incubation times are, therefore, determined empirically for each experiment. All slides are finally washed in distilled water, air-dried at 42°C, and mounted in glycerol jelly. 

Because of the high specificity of an antibody for its epitope, an antibody raised against a particular protein antigen can be used to determine the location of that antigen in a cell using immunofluorescence light microscopy or immuno-electron microscopy. 

Ultrathin sections are picked up on bare nickel or gold grids (copper reacts with OSO4) and incubated, section down, on a drop of antiserum (e.g., on Parafilm or in a microtitre plate) and washed in multiple grid holders. Dilutions of antisera and incubations are as for light microscopy. The primary antibodies, however, are usually incubated for prolonged periods (up to 48 h) at higher dilutions. 

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