Evaluation of nine different fixatives. 1. Preservation of immunoglobulin isotypes, J chain, and secretory component in human tissues.

Eight cross-linking fixatives were tested for their ability to preserve IgG, IgA, IgM and IgD isotypes, J chain, and secretory component (SC) in paraffin-embedded specimens of human tonsils and colonic mucosa. The results were compared with the antigenic preservation afforded by cold 96% ethanol (with or without inclusion of a prefixation 48-hr washing period). A semiquantitative immunofluorescence scoring system was applied. In relation to the cytoplasmic scores of 3.0 assigned to IgG and IgA immunocytes after ethanol fixation, the following median scores were obtained with the other fixatives: routine formalin, 1.3 (IgG and IgA); glutaraldehyde (1%)-formalin, 0.3 (IgG and IgA); Baker's formol calcium, 2.3 (IgG) and 1.5 (IgA); formol sublimate, 1.0 (IgG) and 1.2 (IgA); acetic acid (2%)-formol saline, 2.0 (IgG and IgA); Bouin's fluid and Susa fixative, 0 (IgG) and 1.3 (IgA); and carbodiimide, 2.3 (IgG) and 2.0 (IgA). Most aldehyde-based fixatives afforded poorer result for IgA immunocytes when an anti-alpha-chain reagent of restricted specificity was applied. The result was usually slightly better for IgM cells than for IgG cells, but IgM did not resist fixation in Bouin's fluid and Susa fixative to the same extent as IgA. IgD immunocytes were poorly revealed except after fixation with Baker's formol calcium, carbodiimide, and acetic acid-formol saline, which afforded median fluorescence scores of 1.0-1.8 compared with 3.0 after ethanol fixation. J chain in IgA immunocytes was most intensely stained in sections of ethanol-fixed tissue denatured in acid urea (median score, 3) but was also well revealed after fixation with formalin, acetic acid-formol saline, Bouin's fluid or Susa fixative (median scores, 1.8-2.0). The results with most of the cross-linking fixatives were much less favorable for IgA, IgM, J chain and SC in colonic crypt epithelium than in immunocytes. Only carbodiimide afforded fluorescence scores for IgA and SC approaching those obtained after ethanol fixation (2.0 and 1.5 vs. 3.0 and 2.8). The effect of proteolytic unmasking depended on the fixative - not only for different antigens and locations in the tissue, but also for the same antigen in apparently similar cells.