Standardization of various applications of methacrylate embedding and silver methenamine for light and electron microscopy immunocytochemistry

Abstract

The use of butyl-methyl-methacrylate embedding and the application of the silver methenamine (SM) method as a poststaining of the immunoperoxidase-DAB (IP) procedure led to the standardization of several useful methods for the visualization of tissue antigens at the light and electron microscope level. These procedures included: 1) Standardization of the actual methacrylate embedding; 2) The IP-SM method with and without periodic acid oxidation, which provided 100% intensification of the IP staining; 3) The IP-SM method made it possible to stain semithin sections (0.5 micron), and this in turn, permitted a) clear visualization under the light microscope of the intracellular distribution of antigens and, b) staining, in several adjacent sections, of roughly the same cytoplasmic region of the same cell with different primary antisera; 4) a double immunostaining whereby the first antigen in the sequence was revealed by the IP-SM method and the second by the IP procedure; 5) standardization of the IP and the IP-SM methods for post-embedding staining of ultrathin methacrylate sections. The combined application of methacrylate embedding and the IP-SM, and the use of an appropriate fixative, resulted in an ultrastructural immunocytochemical procedure characterized by a good immunoreactivity of the tissue sections, a strong and selective immunoreaction and a well preserved ultrastructure.

Altered Neuronal Mitochondrial Coenzyme A Synthesis in Neurodegeneration with Brain Iron Accumulation Caused by Abnormal Processing, Stability, and Catalytic Activity of Mutant Pantothenate Kinase 2

“…For immunoelectron microscopy, selected brain sections were labeled with antibody PanK2 549 (1:100) and sequentially incubated with a biotinylated goat anti-rabbit antibody and HRP-conjugated streptavidin. After chromogen reaction with diaminobenzidine (DAB)-containing medium, reaction products were enhanced by the silver methenamine method as described previously (Rodriguez et al, 1984). Sections were postfixed with 1.5% glutaraldehyde and 1% osmium tetraoxide, dehydrated in graded ethanols, and embedded in Epon.…”

Axon pathology in Parkinson’s disease and Lewy body dementia hippocampus contains α-, β-, and γ-synuclein

“…To do this, sections were washed in PBS, blocked with 2% horse serum, and incubated with primary antibody overnight at 4°C after which they were washed with PBS, incubated with biotinylated anti-mouse IgG (Vector Laboratories) for 1 hr, washed again, and then developed by using the avidin-biotin complex method (Vector Laboratories) with diaminobenzidine as a chromogen. Adjacent sections were further processed with a silver enhancement procedure as described (25). Control sections were processed in parallel with the exception of the primary antibody.…”

Identification, cellular and subcellular distribution of 21 and 72 kDa proteins (tuberalins?) secreted by specific cells of the pars tuberalis

“…Sagittal and frontal serial sections, 4 µm thick, were mounted on separate slides. Some PT explants and bovine PT were fixed by immersion in Bouin's fluid and fragmented into blocks of about 1 mm thick; these were dehydrated in methanol and acetone and embedded in butyl-methyl-methacrylate (Rodríguez et al 1984). Serial 1 µm methacrylate sections were individually mounted on separate slides.…”

“…After washing in the same buffer for 30 min, the tissue blocks were fixed in 0·5% OsO 4 in 0·1 M phosphate buffer, pH 7·4, for 2 h. Subsequently, the tissues were washed with distilled water for 15 min, dehydrated and embedded in butyl-methylmethacrylate (Rodríguez et al 1984). Ultrathin sections were stained with uranyl acetate and lead citrate.…”

“…For the immunoperoxidase procedure, sections were run through the following steps: (1) incubation in the primary antibody (Ab-72, diluted 1:1000; Ab-21, diluted 1:1000; antih LH, diluted 1:1000), at room temperature, for 24 h; (2) washes in Tris buffer, pH 7·8; (3) incubation in the second antibody (dilution 1:50), for 5 min; (4) washes in Tris buffer, pH 7·8; (5) incubation in PAP (dilution 1:75), for 5 min; (6) washes in Tris buffer, pH 7·8; (7) incubation in the DAB solution prepared as indicated above, 1 min; (8) washes in distilled water, 15 min. Then, the sections were run through the silver methenamine procedure that allowed visualisation of the antibody-binding site by the presence of fine and densely packed silver grains (Rodríguez et al 1984). Finally, the sections were contrasted with lead citrate.…”