Cell labeling fixation prior

Permeabilization of cells with 0.1-0.2% detergent after paraformaldehyde fixation can leave an uneven cytoplasmic distribution of the labeled proteins, and some of the larger proteins are redistributed to the nuclei. Extraction with 1 % de tergent prior to fixation removes most, but not always all of the exogenous proteins from the cell remnants. 

Figure 5.10. Accumulation of a radiolabelled LMWP in the lysosomes of the proximal tubular cell. Electron microscope autoradiography of renal proximal tubular cells from a rat injected i.v. with [1251]-tyramine-cellobiose-labelled cytochrome-c, 4 h prior to fixation throngh the abdominal aorta. An intense lysosomal accumulation of the protein is observed in three dark electron-dense lysosomes. A few grains are seen over the apical endocytic apparatus. Part of the luminal brush border is found in the upper right hand corner. Magnification, x 25 000. Unpublished data from E. I. Christensen, Arhus, Denmark, and M. Haas, Groningen, Netherlands.

Where a cell surface antigen is of interest, and particularly where the cells are grown in vitro, a labelling method which avoids all the problems of fixation and embedding can be used. Antibody and conjugate incubations can all be completed prior to fixation and embedding for TEM or critical point drying for SEM. 

Phospholipids occur in cells mostly as components of cellular membranes in which they are in close structural relation to proteins. This interaction is an important factor in relation to the problem of phospholipid preservation during dehydration and embedding of tissues. Data on total phospholipid retention, following labeling with fatty acids are given by Kom and Weisman (1966) in the amoeba and by Dermer (1968) in rat intestine. The former have noted better retention of phospholipids than of neutral lipids, while the latter reported that both were retained to the same extent Very little loss of phospholipids, determined chemically, from erythrocyte stroma fixed with glutaraldehyde and osmium tetroxide in the presence of Ca ions was reported by Mitchell (1969). The loss increased from 2 to 7% if the stroma was stored for 7-10 days prior to fixation. 

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