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keep a specimen in the frozen state, and produce frozen sections more suitable for

immunolabelling (Section 4.2.3).

Prior to sectioning, the tissue is usually treated with a chemical agent called a

fixativeto preserve it. Popular fixatives include formaldehyde and glutaraldehyde,

which act by cross-linking proteins, or alcohols, which act by precipitation. All of

these fixatives are designed to maintain the structural integrity of the cell. After

fixation the specimen is usuallypermeabilisedin order to allow a stain to infiltrate

the entire tissue. The amount of permeabilisation (time and severity) depends upon

several factors; for example, the size of the stain or the density of the tissue. These

parameters are found by trial and error for a new specimen, but are usually available

in published protocols. The goal is to infiltrate the entire tissue with a uniform

staining.

4.2.3 Contrast in the light microscope

Most cells and tissues are colourless and almost transparent, and lack contrast when

viewed in a light microscope. Therefore to visualise any details of cellular components

it is necessary to introduce contrast into the specimen. This is achieved either by

optical means using a specific configuration of microscope components, or by

staining the specimen with a dye or, more usually, using a combination of optical

and staining methods. Different regions of the cell can be stained selectively with

different stains.

Optical contrast

Contrast is achieved optically by introducing various elements into the light path of

the microscope and using lenses and filters that change the pattern of light passing

Table 4.2Generalised indirect immunofluorescence protocol

1. Fix in 1% formaldehyde for 30 min


2. Rinse in cold buffer


3. Block buffer


4. Incubate in primary antibody e.g. mouse anti-tubulin


5. Wash 4in buffer


6. Incubate in secondary antibody e.g. fluorescein-labelled rabbit anti-mouse


7. Wash 4in buffer


8. Incubate in anti-fade reagent e.g. Vectashield


9. Mount on slide with a coverslip


10. View using epifluorescence microscopy

109 4.2 The light microscope