A focus on fixation.
Three fixation issues related to immunostaining are discussed here: 1) Generally, a tissue block is fixed, then embedded and sectioned (pre-fixation). The type of fixative applied, crosslinking or coagulating, has an impact on selecting an epitope retrieval method. Individual antigens have a fixation-retrieval characteristic. 2) A long fixation time, especially with crosslinking fixatives, may compromise the result of immunostaining. This negative effect varies among different antigens and can be partially restored by applying a more sensitive/efficient detection system such as tyramide amplification. 3) Sections cut from a fresh frozen tissue block usually are acetone fixed(post-fixation). This was accepted as the "gold standard" for a long time. Post-fixation, however,may have serious consequences for preservation of small peptides leaking from the cut open cells,whereas this is not the case with pre-fixed intact cells. Consequently, the concept of an acetone post-fixed cryostat tissue section as "gold standard" no longer exists and a more appropriate use of the terms immunohistochemistry and immunocytochemistry therefore seems justified. For many antibodies, it is not known whether a formalin fixed, paraffin embedded tissue specimen is appropriate. Suggestions are made for creating a positive control cell block for testing such antibodies.
van der Loos
C M
CM
Department of Pathology, Academic Medical Center M2-230, Meibergdreef 9, NL-1105 AZ Amsterdam, The Netherlands. c.m.vanderloos@amc.uva.nl
D003432:Cross-Linking Reagents; D015925:Cryopreservation; D000939:Epitopes; D005557:Formaldehyde; D006801:Humans; D007150:Immunohistochemistry; D014066:Palatine Tonsil / Q000033:anatomy & histology / Q000378:metabolism; D016707:Tissue Fixation
D003432:Cross-Linking Reagents; D000939:Epitopes; D005557:Formaldehyde