Protein A‐gold electron microscopic immunocytochemistry: Methods, applications, and limitations

Abstract

The postembedding protein A-gold immunocytochemical approach has been introduced as a n alternative to other techniques for the ultrastructural localization of antigenic sites. The present review deals with the development, the theoretical background, and technical approach of the protein A-gold method as well as the different modifications introduced in order to enhance the resolution of the results and to perform double labelings on the same section. Various examples demonstrate the reliability and the wide range of application of this technique. In addition, some problems, pitfalls, and limitations particular to this method are reported.

Alteration in the distribution of type IV collagen in glomerular basal laminae in diabetic rats as revealed by immunocytochemistry and morphometrical approach

“…For the study of renal tissue at the electron microscope level, small fragments of the renal cortex were fixed by immersion in 0.1 mol/1 phosphate buffer freshly prepared 4% formaldehyde (pH 7.4) for 2 h at 4~ and embedded in Lowicryl K4M (Chemische Werke Lowi GmbH Waldkraiburg, FRG) at -20 ~ as described previously [25]. These conditions of fixation and embedding were found to be optimal for the ultrastructural localization of type IV collagen by the protein A-gold immunocytochemical technique [31].…”

“…For the immunocytochemical study of type IV collagen, specific antibodies provided by Dr. G. Martin (National Institute of Health, Bethesda, Maryland, USA) and the protocol for the protein A-gold technique [25] were used. These antibodies were raised in sheep using an antigen extracted from the mouse EHS tumor and purified by salt precipitation and ion exchange chromatography [33,34]; they react with basal laminae of various animal tissues and in particular with rat glomerular basal laminae [16,18], and their high specificity has been demonstrated previously using several approaches [16,18,35].…”

“…These antibodies were raised in sheep using an antigen extracted from the mouse EHS tumor and purified by salt precipitation and ion exchange chromatography [33,34]; they react with basal laminae of various animal tissues and in particular with rat glomerular basal laminae [16,18], and their high specificity has been demonstrated previously using several approaches [16,18,35]. To locate type IV collagen, the protein A-gold immunocytochemical postembedding approach was applied [25]. In short, the thin sections of renal tissue were first incubated with the specific antibody at 88 dilution in phosphate buffer for 2 h at room temperature and after rinsing, the sections were further incubated for 30 min with the protein A-gold complex.…”

“…In short, the thin sections of renal tissue were first incubated with the specific antibody at 88 dilution in phosphate buffer for 2 h at room temperature and after rinsing, the sections were further incubated for 30 min with the protein A-gold complex. This complex was prepared using gold particles of 14 nm diameter [25] and to enhance the contrast for viewing the gold particles at low magnification, the sections were only lightly stained with uranyl acetate. Tissues from normoglycaemic and hyperglycaemic animals were processed in parallel.…”

“…Recently however, some studies have detected biochemical alterations in the proteoglycan component of 'diabetic' basal laminae [22][23][24]. Consequently, we are studying the ultrastructural localization of the different components of the basal lamina in renal tissues from normal and streptozotocin-diabetic rats by applying the high resolution protein A-gold immunocytochemical technique [25,26]. Our first step, which is reported in the present study, was to localize type IV collagen.…”