Some fixation reagents reduce or abolish the detectability of Ia-antigen and HLA-DR on cells.
The effect of paraformaldehyde (PF), glutaraldehyde (GT), methanol (ME), ethanol (ET) and acetone (AC) fixation on the detectability of Ia antigens on murine and rat peritoneal exudate (PE) and resident peritoneal (RP) macrophages (M phi), and on detectability of HLA-DR antigens on human blood leukocytes (HBL) and human splenic M phi (HSM phi) was examined. Ia-antigen on Mø from H-2k mice was detected by a rosetting assay using erythrocytes (E) to which a monoclonal antibody (MoAb) reactive to Ia.2 (E anti-Ia.2) had been coupled, and by the direct binding of 125I-labeled anti-Ia.2. The antigen was detected on Wistar/Furth (W/Fu) rat RPMø splenocytes (SC) by rosetting with E coupled with a MoAb to the murine determinate Ia.17, which cross-reacts with an Ia-like molecule on cells from the W/Fu strain. HLA-DR framework determinants were detected on HBL and HSMø by the binding of 125I-labeled MoAb and by an avidin-biotinylated peroxidase procedure. Exposure of murine PEMø or RPMø to 1% PF or 0.5% GT for 15 min at room temperature reduced 125I-anti-Ia.2 binding and E anti-Ia.2 rosetting by at least 60%; the radioimmunoassay was more affected by the fixatives than was the rosetting assay. Further, PEMø were more sensitive to the effect of PF fixation than were RPMø. Treatment of freshly isolated RPMø with 1% PF reduced the proportion of Ia-bearing cells detected by the rosetting assay by greater than 50%. Culturing alone did not affect the detectability of Ia on RPMø as assessed by the rosetting test, but cultured RPMø were more sensitive to the effects of FX fixation than fresh cells except when lymphokine from Con A-stimulated murine SC was included in the culture medium. Similar losses of HLA-DR were recorded when HBL and HSMø were exposed to PF, GT, ME or ET, but brief (less than 20 s) treatment with cold AC did not appreciably reduce antigen detectability. Procedures in which fixation takes place after the primary antibody binding step did not result in an appreciable loss of detectable Ia. Thus, commonly used fixatives affect the detectability of Ia and Ia-like antigens on a variety of cells. Results obtained from assays on cells treated prior to the primary antibody binding step, therefore, must be interpreted with caution.
Walker, WS; Beelen, RH; Buckley, PJ; Melvin, SL; Yen, SE
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