Double autoimmunostaining with glycine treatment.

Double autoimmunostaining by a sequential twice-repeated enzyme-labeled polymer method was examined on archival paraffin sections of formalin-fixed human tissue using an autoimmunostaining apparatus to determine optimal conditions for glycine treatment, to select the best combination of dyes for the horseradish peroxidase-hydrogen peroxide reaction, and to investigate mounting methods for preparing permanent specimens. The optimal glycine treatment determined by changing the incubation time in 0.1 M glycine hydrochloride buffer, pH 2.2, was glycine buffer washing three times for 1 min each, with suppression of nonspecific binding of the primary antibody by protein blocking. Combinations of DAB and AEC, SG and AEC with Ultramount, and DAB and VIP or NovaRED and SG with the VectaMount were found usable for the double autoimmunostaining, based on color analysis of the dyes. Pairs of primary antibodies, CD68 and anti-fascin antibodies CD3 and CD79a, and anti-Ki-67 antigen and anti-p53 antibodies were applicable in double autoimmunostaining with appropriate antigen retrieval for each pair of primary antibodies. Consequently, good sequential double autoimmunostaining should include masking the nonspecific binding of primary antibodies, optimal glycine treatment, and selection of adequate dyes and mounting methods.

Improvement of supersensitive immunohistochemistry with an autostainer: a simplified catalysed signal amplification system.

The ImmunoMax/catalysed signal amplification (CSA) system is a supersensitive method of paraffin immunohistochemistry. It incorporates antigen retrieval, the streptavidin-biotin complex (sABC) method, and the catalysing reporter deposition/catalysing biotinylated tyramide reaction. Strong, non-specific cytoplasmic reaction in the ImmunoMax/CSA is due to endogenous biotin unmasked in the antigen retrieval step. We examined procedures to diminish this non-specific immunoreaction and improved the ImmunoMax/CSA. Antigen retrieval in a hot water bath yielded a smaller endogenous biotin immunoreaction than antigen unmasking in an autoclave. Post-antigen retrieval fixation in buffered 10% formalin solution suppressed the biotin immunoreaction but masked the target antigen, Ki67. Post-reaction washing with 0.1% Tween 20 in Tris-HCl buffer at 35 degrees C did not diminish the endogenous biotin immunoreaction. Animal serum also did not suppress the non-specific immunoreactivity of biotin and antibodies. Because endogenous biotin is detected by duplicated biotin-streptavidin reactions in the ImmunoMax/CSA, we replaced the sABC step with a labelled polymer secondary antibody (the EnVision system)--a simplified CSA system--because the sensitivity of the EnVision system was the same as that of the sABC method. The non-specific immunoreaction induced by the EnVision system was masked competitively by blocking protein. By using an antibody against Ki67 antigen that can react only with the nucleus, we were able to evaluate the non-specific cytoplasmic immunoreaction induced by the detection system. We believe that the simplified CSA system will open up the field of supersensitive paraffin immunohistochemistry.