A method for preparing and staining histological sections containing titanium implants for light microscopy

An improved method for preparing and staining ground tissue-implant sections for light microscopy is presented. Undecalcified tissue blocks with titanium implants were dehydrated in an ascending series of ethanol and stained in toto with basic fuchsin. Specimens were infiltrated and embedded in methyl methacrylate and sections were prepared using a cutting-grinding-system. The polished surface was counterstained with light green or anilin blue. Light polymerizing resin was used as slide mounting medium and for mounting the coverglass. The sections obtained were 10-15 microns thick with tissue architecture which clearly differentiated structures at the tissue-implant interface. The method was very useful for computer assisted morphometric analysis.     

A ruthenium red-toluidine blue procedure for staining epoxy sections in the light microscopy.

Ruthenium red (RR) has been widely used as a fixation additive for electron microscopy on the basis of its capacity to retain acid mucopolysaccharide residues of the cell surface coat. Little is known about the properties of this compound as a direct staining agent for epoxy-resin embedded material. In this study, semithin sections of Epon-infiltrated muscle tissue samples have been treated with 1% RR followed by counterstaining with 1% toluidine blue. This 2 step staining procedure has proven to be simple, rapid, and reliable and to give a dramatic improvement in image resolution and contrast. Thus, we believe that histochemical procedures employing RR may find in the future interesting applications for the direct staining of epoxy-resin embedded tissues.     

Immunogold silver staining for light microscopy

 The immunogold silver staining method (IGSS) is widely used as a sensitive and specific immunohistochemical visualisation technique. IGSS involves the specific deposition of metallic silver at the site of immunogold labelling and provides a means of visualisation at low magnification by light or electron microscopy. Silver developers for IGSS rapidly deposit metallic silver only at the site of heavy metals, including gold and silver, because of their catalytic activity. The developing solution contains the silver ions and reducing agent necessary for this reaction. Using different silver salts as ion donors and by selecting an appropriate temperature and pH, visible amounts of silver can be deposited in a few minutes at the site of colloidal gold labelling while little non-specific background deposition occurs. Inclusion of protective colloids in the solution can also be used to control the reaction. Although studies of the chemical basis of silver deposition around unlabelled colloidal gold date back to 1939, immunogold enhancement by silver was established in 1983. The IGSS method evolved from the combination of disparate photographic, histochemical and immunogold techniques which have been effectively combined and optimised over the last 10 years to provide a visualisation system which is well suited to many immunohistochemical studies. Accepted: 29 April 1996     

Bismuth staining for light and electron microscopy.

Bismuth salts on aldehyde fixed tissue give a highly selective pattern of staining suitable for light and electron microscopy. Structures stained include the nucleolus, ribosomes, inter- and perichromatin granules, the Golgi complex beads and the outer face of the tubule doublets of mouse sperm, certain neurosecretory vesicles believed to contain biogenic amines, some junctions (some central synapses, neuromuscular junctions, tight junctions), specialized membranes such as the post acrosomal dense lamina of mouse sperm and the inner alveolar membrane of Paramecium, and a variety of structures associated with the cytoplasmic face of membranes, such as plasma membrane plaques, cleavage furrows, the leading edge of the spreading acrosome and sperm annuli.Staining is not reduced by nucleases and spot tests show no reaction between nucleic acids and bismuth under conditions similar to those used to stain tissues. However, spot tests do show strong binding of bismuth by basic proteins and by some phosphorylated molecules.It is hypothesized that bismuth reacts with cell components in two ways, distinguishable by their glutaraldehyde sensitivity. For example, staining of the nucleolus and ribosomes is blocked by glutaraldehyde but the inter- and perichromatin granules and the GC beads are unaffected. Spot tests show that basic proteins (histones, protamines, polylysine and polyargenine) and other molecules with free amino groups (5HT, tryptamine, dopamine) bind bismuth strongly, a reaction that is blocked to varying degrees by glutaraldehyde. We presume that most bismuth staining of tissues is due to reaction with amine groups and is glutaraldehyde sensitive and some may be due to guanidine groups which are less sensitive to fixation by glutaraldehyde. Organic phosphates may be the cause of the glutaraldehyde insensitive staining since ATP and some other phosphates bind bismuth in a reaction that is not blocked by glutaraldehyde.     

Observations on the selective demonstration of nucleolar material by protein staining techniques in epon thick sections

Summary The effect of different protein stainings on thick sections of Epon embedded salivary glands of Chironomus pallidivittatus is described. Pretreatment of sections with several metallic salts affected the affinity for aniline blue black in different structures. The uranyl and aluminium ions followed by aniline blue black resulted in a highly selective nucleolar staining which proved also efficient in Allium cepa meristematic cells and which seems useful for the preferential demonstration of nucleolar material in thick sections.This work was supported by grants from Deutscher Akademischer Austauschdients to S.A.B. and from Alexander von Humboldt-Stiftung to J.C.S.     

Observations on the selective demonstration of nucleolar material by protein staining techniques in epon thick sections.

The effect of different protein stainings on thick sections of Epon embedded salivary glands of Chironomus pallidivittatus is described. Pretreatment of sections with several metallic salts affected the affinity for aniline blue black in different structures. The uranyl and aluminium ions followed by aniline blue black resulted in a highly selective nucleolar staining which proved also efficient in Allium cepa meristematic cells and which seems useful for the preferential demonstration of nucleolar material in thick sections.