Immunochemistry is the identification of a certain antigen in a histological tissue section or cytological preparation via an antibody specific to the antigen. The localization of the primary antibody (and therefore the target antigen) is then visualized microscopically via an appropriate enzymatic or fluorescent detection system. There are numerous techniques available, depending on the complexity and on the degree of sensitivity required.
To set the scene, there are basically five types of specimen commonly used in immunochemical staining experiments:
• Paraffin-embedded tissue sections.
• Frozen tissue sections.
• Free-floating tissue sections.
• Cytological specimens as traditional smears.
• Cytological specimens as monolayer preparations (including cytospin preparations).
Paraffin-embedded sections are probably the most common type of specimen to be immunochemically stained. Tissue blocks are obtained from a suitable gross specimen, fixed to preserve morphology, and then processed to paraffin wax in order to give the tissue support during microtomy. Sections are cut at around 4 μm thickness and floated out in a water bath before being picked up on to a glass microscope slide. The sections are then dried at around 60°C in order to increase their adherence to the slide and to help to iron out any creases obtained during microtomy or floating. Before immunochemical staining can occur, sections must be dewaxed and any necessary pre-treatments performed.
Frozen tissue sections tend to be utilized when a rapid diagnosis is required or when certain antigens need to be visualized that are sensitive to aldehyde fixation and tissue processing. Generally, frozen sections allow much better antigen preservation than paraffin sections and are favored by some researchers due to the absence of an excessive fixation and processing regime, leaving antigens in a more 'native' form. Markers for estrogen receptors used to fall into this category until antibodies were developed that detected an epitope present in formaldehyde-fixed, paraffin-embedded sections. When prepared correctly, tissue morphology in frozen sections is of a reasonably high quality, but does not appear to compare with that of paraffin sections, probably due to the higher degree of fixation in paraffin sections. Sectioning of frozen tissue is notably more technically demanding, especially if the tissue is calcified or has a high lipid content. To prepare a frozen section, fresh tissue is plunged into a suitable cryo-agent such as liquid nitrogen and once frozen is placed on to a cryostat chuck and embedded in a suitable embedding medium such as OCT (optimal cutting temperature). Once the embedding medium has set, 4 μm sections are obtained by cryotomy and mounted on to a glass microscope slide.
Free-floating sections are commonly favored by those working on sensitive neurological antigens due to the speed at which a result can be obtained.
Following cardiac perfusion with an aldehyde-based fixative, tissue is removed from the gross specimen and sectioned, typically at around 50 μm thickness. Sections are then floated out on to a water bath and immunochemically stained in situ. The thickness of the specimen allows this to be performed, although particular attention must be paid to washing steps in order to reduce nonspecific background staining due to antibodies becoming 'trapped' in the thick tissue.
Cytological specimens are typically in the form of a conventional smear or as a monolayer obtained via automated technology (for example, Thinprep) or grown on a glass coverslip. Nonspecific background staining can be a problem with cytological specimens, since the clustering of cells can act to 'trap' antibodies. However, this artifact tends to be of less intensity than true positive staining.
It must be stressed that it is the aim of every immunochemical laboratory to obtain reproducible and accurate results every time. There are a plethora of other parameters that have significant effects on the outcome of immunochemical staining. It is common to find scrutiny concerning the reagents and protocols used during an immunochemical staining experiment, for instance reagent concentrations, incubation times, and blocking steps; but in actual fact the results are being predisposed from the point of specimen collection onwards. An equal amount of notice should be paid to how the specimen is treated before being subjected to immunochemical staining as to the staining procedures themselves.