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Tissue preparation is the cornerstone of immunohistochemistry. To ensure the preservation of tissue architecture and cell morphology, prompt and adequate fixation is essential. However, inappropriate or prolonged fixation may significantly diminish the antibody binding capability.
There is no one universal fixative that is ideal for the demonstration of all antigens. However, in general, many antigens can be successfully demonstrated in formalin-fixed paraffin-embedded tissue sections. The discover and development of antigen retrieval techniques further enhanced the use of formalin as routine fixative for immunohistochemistry in many research laboratories.
For best results, vertebrate tissues (especially neuronal tissues) usually require fixation by transcardial perfusion for optimal tissue preservation. The most common fixatives used for immunohistochemistry are the followings:
a) 4% paraformaldehyde in 0.1M phosphate buffer
b) 2% paraformaldehyde with 0.2% picric acid in 0.1M phosphate buffer
c) PLP fixative: 4% paraformaldehyde, 0.2% periodate and 1.2% lysine in 0.1M phosphate buffer
d) 4% paraformaldehyde with 0.05% glutaraldehyde (TEM immunohistochemistry)
Some antigens will not survive even moderate amounts of aldehyde fixation. Under this condition, tissues should be rapidly fresh frozen in liquid nitrogen and cut with a cryostat without infiltrating with sucrose. The sections should be kept frozen at -20 C or lower until fixation with cold acetone or alcohol. After fixation, the sections can be processed using standard immunohistochemical staining protocols
Since its introduction, paraffin wax has remained the most widely used embedding medium for diagnostic histopathology in routine histological laboratories. Accordingly, the largest proportion of material for immunohistochemistry is formalin-fixed, paraffin-embedded. Paraffin sections produce satisfactory results for the demonstration of majority of tissue antigens with the use of antigen retrieval techniques.
Certain cell antigens do not survive routine fixation and paraffin embedding. So the use of frozen sections still remains essential for the demonstration of many antigens. However, the disadvantage of frozen sections includes poor morphology, poor resolution at higher magnifications, special storage needed, limited retrospective studies and cutting difficulty over paraffin sections.
Vibratome sections have some advantages when doing immunohistochemistry since the tissue is not processed through organic solvents or high heat, which can destroy the antigenicity. In addition, the morphology of tissue sections is not disrupted due to no freezing and thawing needed. Vibratome sections are often used for floating immunostaining, especially for pre-embedding EM immunohistochemistry. The disadvantage of vibratome sections is that the sectioning process is slow and difficult with soft and poorly fixed tissues. In addiction, the chatter marks or vibratome lines are often appeared in the sections.
Small blocks of tissue (less than 5 mm thick) can be processed as whole mounts. The advantage of whole mount preparations is that the results provide three dimensional information about the location of antigens without the need for reconstruction from sections. However, the major limitation of using whole mounts is antibody penetration may not be complete in the tissue, resulting in uneven staining or false negative staining. So Triton X-100 or saponin treatment are used routinely for whole mount immunohistochemistry to enhance penetration of the antibody.