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

• Published in: Journal of neuroscience methods 2001-11, Vol.112 (1), p.43-49
• Main Authors: Freedman, Lorin J., Maddox, Marcelia T.
• Format: Article
• Language: English
• Subjects: Animals; Antibodies; Avidin; Avidin - analogs & derivatives; Axons - metabolism; Axons - ultrastructure; Biological and medical sciences; Biotin; Biotin - analogs & derivatives; Carrier Proteins - metabolism; Double-bridge; Female; Fundamental and applied biological sciences. Psychology; General aspects. Models. Methods; Gyrus Cinguli - cytology; Gyrus Cinguli - metabolism; Horseradish Peroxidase; Immunohistochemistry; Immunohistochemistry - methods; Macaca mulatta; Male; Membrane Glycoproteins - metabolism; Membrane Transport Proteins; Nerve Tissue Proteins; Serotonin - metabolism; Serotonin Plasma Membrane Transport Proteins; Tyramide signal amplification; Tyramine - analogs & derivatives; Vertebrates: nervous system and sense organs
• ISSN: 0165-0270; 1872-678X
• DOI: 10.1016/S0165-0270(01)00454-X

Often it is difficult to detect very small amounts of antigen with conventional immunohistochemical techniques. We evaluate three amplification techniques involving anti-biotin or anti-avidin double-bridges or biotinylated tyramide amplification to enhance the sensitivity of serotonin transporter immunohistochemistry. 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–peroxidase complex (ABC). For the biotinylated anti-avidin technique, after the ABC, 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 peroxide, followed by another incubation in ABC. The anti-biotin double-bridge also resulted in a large increase in the number of stained fibers and the intensity of labeling with no increase in background. A biotinylated anti-avidin double-bridge also produced significant signal amplification but significant background. The biotinylated tyramide technique resulted in an even larger increase in the number of labeled fibers and an intensity of their staining with a moderate amount of background staining. However, this advantage was not present at high dilutions of primary antibody. Thus, the anti-biotin double-bridge is likely to be useful in immunohistochemistry and immunofluorescence as well as other situations where increased sensitivity and low background from biotin markers is needed. The biotinylated tyramide technique may also be useful where some degree of background labeling is acceptable.