Figure 1.

Effects of 100% ethanol treatment on 3-amino-9-ethylcarbazole (AEC)–derived signals and of microwave (MW) treatment on horseradish peroxidase (HRP) activity and antigenicity of primary antibodies. All sections were stained with mouse anti–glial fibrillary acidic protein (GFAP) antibodies (1:10,000), HRP-conjugated anti-mouse antibodies (DAKO, K4000), and AEC according to the first staining step described in Materials and Methods (A). Subsequently, the same sections were directly subjected to MW treatment, destained with 100% ethanol (B), and dipped in DAB staining solution (C). The other AEC-stained sections (D) were subjected to MW treatment and alcohol treatment, incubated again with HRP-conjugated secondary antibody (DAKO, K4000), and developed by DAB for 10 min (E). When sections were processed as other sections (E) had been done, except that excess primary antibody was applied (1:1000) in the first round of staining, unwanted cross-reactive signals were detected (F). Bar = 50 µm.