Immunolabelling and visualization in living cells

Immrmolabelling of organelles or structures by fluorescent antibodies in living cells has the advantage, compared to work in fixed cells, that the dynamics of the labelled structures or molecules can be studied by for example time-lapse microscopy. Also, labelling in living cells does not suffer from possible problems induced by the fixation or permeabilization of the cells as is necessary for immimofluorescence or electron microscopy. 

A number of approaches to fluorescently label cytoplasmic and nuclear structures or organelles in living cells are presently available. They vary considerably in terms of ease of use, how specifically they label the target molecules, and in how much they interfere with cellular function. Direct microinjection of fluorescently labelled antibodies into living cells is a universal and efficient way of labelling cellular targets in vivo. In order to avoid interference with cellular function or non-specific cross-reactions the fluorescent antibodies should be prepared as described in Section 2.1 and be first characterized in vitro before they are introduced into cells. 

Secondly, the injected labelled antibodies should not interfere with cellular function, which could significantly change the distribution of the antibodies antigens in the living cells. 

It is therefore most useful to microinject the fluorescent antibodies over a whole range of concentrations in order to achieve the optimal labelling conditions, but which does not interfere with cell function 

A detailed discussion of suitable microinjection equipment and technical aspects is beyond the scope of this chapter and are described in ref. 3. The glass micropipettes used for penetration of cells and delivery of the sample are avail- 

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