A comparison of anti-biotin and biotinylated anti-avidin double-bridge andbiotinylated tyramide immunohistochemical amplification

Often it is difficult to detect very small amounts of antigen with conventi onal immunohistochemical techniques. We evaluate three amplification techni ques involving anti-biotin or anti-avidin double-bridges or biotinylated ty ramide amplification to enhance the sensitivity of serotonin transporter im munohistochemistry. For the anti-biotin double-bridge, after the secondary antibody, the sections were incubated in anti-biotin antibody followed by a second incubation in the secondary antibody and then avidin-biotin-peroxid ase complex (ABC). For the biotinylated anti-avidin technique, after the AB C, sections were incubated in biotinylated anti-avidin, followed by another incubation in ABC. For the biotinylated tyramide technique, after the ABC step, sections were incubated in biotinylated tyramide and hydrogen peroxid e, followed by another incubation in ABC. The anti-biotin double-bridge als o resulted in a large increase in the number of stained fibers and the inte nsity of labeling with no increase in background. A biotinylated anti-avidi n double-bridge also produced significant signal amplification but signific ant background. The biotinylated tyramide technique resulted in an even lar ger increase in the number of labeled fibers and an intensity of their stai ning with a moderate amount of background staining. However, this advantage was not present at high dilutions of primary antibody. Thus, the anti-biot in double-bridge is likely to be useful in immunohistochemistry and immunof luorescence as well as other situations where increased sensitivity and low background from biotin markers is needed. The biotinylated tyramide techni que may also be useful where some degree of background labeling is acceptab le. (C) 2001 Elsevier Science B.V. All rights reserved.