Evaluation of nine different fixatives. 2. Preservation of IgG, IgA and secretory component in an artificial immunohistochemical test substrate.
An artificial substrate was developed for quantitative testing of the ability of various fixatives to preserve the reactivity of IgG and IgA isotypes (gamma and alpha chains) and the secretory component (SC) of secretory IgA as model antigens. Polymerized normal rabbit serum was used as matrix and defined amounts (10-0.1 g/l) of antigen were incorporated into it by diffusion before fixation and paraffin embedding. The various fixatives comprised alcohol, routine formalin, glutaraldehyde(1%)-formalin, Baker's formol calcium, formol sublimate, acetic acid(2%)-formol saline, Bouin's fluid, Susa fixative, and carbodiimide. The detection sensitivity afforded by these fixatives was defined as the immunofluorescence staining end point. Compared to the reference value obtained with alcohol (gamma and alpha chains, 0.06 g/l of IgG and IgA; SC, 0.12 g/l of colostral IgA), an antigen concentration at least 8 times higher was necessary for detection with most of the cross-linking fixatives. Bouin's and Susa fixatives were peculiar in that they required more than 150 times higher antigen concentration for detection of IgG but only 3-8 times higher for IgA. The determined sensitivities were compared with the immunofluorescence performance results obtained on human tissues prepared with the same fixatives; excepting carbodiimide (which produced unacceptable autofluorescence of the substrate matrix) a remarkably good correlation was found with regard to IgG- and IgA-producing cells (especially of the former isotype) and secretory epithelium (IgA and SC). However, the latter result depended on pronase treatment of the tissue sections to unmask epithelial antigens.(ABSTRACT TRUNCATED AT 250 WORDS)
D000818:Animals; D005069:Evaluation Studies as Topic; D005404:Fixatives; D005455:Fluorescent Antibody Technique; D007120:Immunochemistry; D007070:Immunoglobulin A; D007074:Immunoglobulin G; D007136:Immunoglobulins; D007158:Immunologic Techniques; D011817:Rabbits; D012635:Secretory Component
D005404:Fixatives; D007070:Immunoglobulin A; D007074:Immunoglobulin G; D007136:Immunoglobulins; D012635:Secretory Component