The application of a biotin-anti-biotin gold technique providing a significant signal intensification in electron microscopic immunocytochemistry: a comparison with the ultrasmall immunogold silver staining procedure

Abstract

A three-step biotin-anti-biotin gold-detection system (method A) has been applied for ultraimmunocytochemistry using ultrasmall colloidal gold (0.8 nm) linked to anti-biotin antibodies which were visualized and enhanced by silver reduction. The reactivity for glucagon in human pancreatic islets and for cytochrome-c oxidase in heart mitochondria has been compared to a two-step ultrasmall immunogold technique (method B). For both antigens, method A provided significantly higher labelling indices (P<0.001): the labelling density for cytochrome-c oxidase was 223/microm2 using method A and 78/microm2 using method B. For glucagon, the labelling density was 1455/microm2 with method A and 322/microm2 with method B. The results demonstrate that the silver-intensified biotin-anti-biotin gold-detection system is a valuable immunocytochemical method for signal enhancement. The method utilizes biotinylated antibodies from different species, allowing its broad application at the electron microscopic level.

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“…For ultraimmunocytochemistry, the tissue was fixed in the periodate-lysine-paraformaldehyde fixative, according to McLean and Nakane (19), with 0.5% glutaraldehyde for 24 hours and embedded in Lowicryl K4M (Chemische Werke Lowi, Waldkreiburg, Germany; Polysciences, Warrington, PA, U.S.A.), as previously described (20). As a control, the specific primary antibody was omitted or substituted with nonimmune immunoglobulin G of the same species as the primary antibody (isotype control).…”

“…For ultraimmunocytochemistry, the following immunolabeling procedure was used (20): ultrasections were mounted on nickel grids, washed in phosphate-buffered saline (PBS) (10 mmol/L phosphate buffer, 150 mmol/L NaCl, pH 7.4) for 5 minutes, placed in 50 mmol/L lysine in PBS for 15 minutes to inactivate aldehyde groups after aldehyde fixation, rinsed in PBS/bovine serum albumin (BSA), treated with normal goat serum, 5% in PBS with BSA (5%) for 30 minutes. The primary antibody, diluted in PBS + 0.1% BSA was applied overnight at room temperature in a moist chamber.…”

Nemaline Cardiomyopathy in a Young Adult: An Ultraimmunohistochemical Study and Review of the Literature

“…Postembedding ultraimmunohistochemistry has been performed on deparaf nized heart muscle tissue reembedded in Lowicryl. Sections were treated with 5% normal goat-serum in phosphate-buffered saline ( PBS ) with 5% bovine serum albumin ( BSA ) for 30 min, treated with the primary antibody diluted in PBS ‡ 0.1% BSA overnight at 4 C, washed in PBS, treated with a goat antimouse ultrasmall gold ( 0.8 mm )-labeled antibody in PBS for 2 h. After a xation step in glutaraldehyde ( 2% in PBS ) for 10 min, a wash step in PBS for 5 min, and 4 wash steps in distilled water, silver reduction was preformed as previously described [ 32,33 ].…”

Histochemistry and cell biology: what's in a name?

“…For in situ hybridization in experimental settings, as well as on routine diagnostic material, the use of various haptenized tyramides has proven to be highly advantageous (Paratore et al 1999;Speel 1999;Speel et al 1998;van de Corput et al 1998;Wiedorn et al 1999). For immunoelectron microscopy, a three step biotin-anti-biotin technique, using ultrasmall (0.8 nm) gold particles together with silver intensification, provided significantly higher labeling indices (p<0.001) for glucagon and cytochrome-c oxidase, as compared to the conventional two-step technique (Müller-Hocker et al 1998). …”