A selective stain for mast cells.

A selective stain for mast cells in tissue sections is presented. The procedure is based on the resistance to destaining with absolute ethanol-acetic acid of the complex acid mucopolysaccharide-Toluidine Blue reinforced with ferrioxamine B.     

Lead asparate, an en bloc contrast stain particularly useful for ultrastructural enzymology

Lead aspartate is a new en bloc stain for electron microscopy. Its predictable staining depends on chelation that results from the interaction of the two stain components, lead nitrate and aspartic acid, which must be present in a specific ratio. Lead aspartate stain is 0.02 M in lead nitrate and 0.03 M in aspartic acid, adjusted to pH 5.5. Cells or tissues are stained at 60 degrees C for 30 to 60 min. Cells stained en bloc with lead aspartate closely resemble cells stained on grids by lead citrate, except that the former seldom have contamination. En bloc staining with lead aspartate bypasses the grid-staining step so that samples can be viewed and photographed immediately after they are thin-sectioned. The lower pH of the lead aspartate solution allows counterstaining of enzyme reaction products that dissolve in the highly alkaline lead citrate stain. Lead aspartate en bloc staining to enhance contrast should especially benefit studies of ultrastructure requiring a clean and predictably lead stain.     

Luna stain, an improved selective stain for detection of microsporidian spores in histologic sections

Microsporidia in histologic sections are most often diagnosed by observing spores in host tissues. Spores are easy to identify if they occur in large aggregates or xenomas when sections are stained with hematoxylin and eosin (H&E). However, individual spores are not frequently detected in host tissues with conventional H&E staining, particularly if spores are scattered within the tissues, areas of inflammation, or small spores in nuclei (i.e. Nucleospora salmonis). Hence, a variety of selective stains that enhance visualization of spores is recommended. We discovered that the Luna stain, used to highlight eosinophils, red blood cells, and chitin in arthropods and other invertebrates, also stains spores of Pseudoloma neurophilia. We compared this stain to the Gram, Fite's acid fast, Giemsa, and H&E stains on 8 aquatic microsporidian organisms that were readily available in our 2 laboratories: Loma salmonae, Glugea anomala, Pseudoloma neurophilia, Pleistophora hyphessobryconis, Pleistophora vermiformis, Glugea sp., Steinhausia mytilovum, and an unidentified microsporidian from UK mitten crabs Eriocheir sinensis. Based on tinctorial properties and background staining, the Luna stain performed better for detection of 6 of the 8 microsporidia. Gram stain was superior for the 2 microsporidia from invertebrates: S. mytilovum and the unidentified microsporidian from E. sinensis.     

A rapid and simple staining method, using Toluidine Blue, for analysing mitotic figures in tissue sections

Summary Mitotic index is a clinically important parameter in cancer pathology. We developed a staining method using Toluidine Blue to detect efficiently and rapidly mitotic figures in sections of formalin-fixed paraffin-embedded human and rat tisues. Sections were stained at acid pH with a 0.01% Toluidine Blue solution after removal of RNA with hydrochloric acid or ribonuclease. The optimal pH of the TB staining solution was found to be 4.5 for rat tissues and 3.5 for human tissues. This procedure stained mitotic figures much more intensely than other (extra)cellular structures. A quantitative estimate of the total number of nuclei in the field where mitotic figures were counted, was obtained in an adjacent section hydrolysed in 5 N hydrochloric acid and stained by the Feulgen reaction with a Schiff-type reagent containing 0.01% Toluidine Blue. This method specifically stained interphase and mitotic nuclei and the field cellularity could be quantified by image cytometry. When these procedures were performed on two consecutive serial sections, a mitotic index could be determined accurately by relating the count of mitotic figures to the number of tumour cells.     

A rapid, sensitive histochemical stain for myelin in frozen brain sections.

A staining technique is described whereby frozen brain sections are incubated in a vehicle containing gold chloride. After 2-4 hr in this solution, both large myelinated bundles and fine individually myelinated fibers are darkly stained. Advantages of this technique over conventional myelin stains include speed, sensitivity, metachromatic staining, and compatibility with formalin-fixed and frozen cut sections. Possible histochemical mechanisms are discussed.     

A method for obtaining mitotic figures from blood leucocyte cultures of rainbow trout, Salmo gairdneri

A method for obtaining dividing cells for chromosome analysis from blood cultures of the rainbow trout, Salmo gairdneri , is described. The effects of different phytohaemagglutinin and foetal calf serum concentrations, oxygenation of the cultures, and varying the initial volume of cell inoculum on the number of mitoses obtained are described and discussed.     

Different processing phases for features, figures, and selective attention in the primary visual cortex

Our visual system imposes structure onto images that usually contain a diversity of surfaces, contours, and colors. Psychological theories propose that there are multiple steps in this process that occur in hierarchically organized regions of the cortex: early visual areas register basic features, higher areas bind them into objects, and yet higher areas select the objects that are relevant for behavior. Here we test these theories by recording from the primary visual cortex (area V1) of monkeys. We demonstrate that the V1 neurons first register the features (at a latency of 48 ms), then segregate figures from the background (after 57 ms), and finally select relevant figures over irrelevant ones (after 137 ms). We conclude that the psychological processing stages map onto distinct time episodes that unfold in the visual cortex after the presentation of a new stimulus, so that area V1 may contribute to all these processing steps.     

Bolton-Hunter reagent as a vital stain for developing systems

The Bolton-Hunter reagent, N-succinimidyl 3-(4-hydroxy, 5-[¹²⁵I]iodophenyl)propionate, was used as a vital stain for developing amphibian and tunicate embryos and for isolated cells (human erythrocytes and cultured chick limb mesenchymal cells). We found that the Bolton-Hunter reagent can be used on living cells at room temperature with techniques that are quite similar to the techniques routinely used to label isolated macromolecules in vitro. At concentrations of vital stain that were sufficient to label intracellular proteins in intact-cells, labeled cells underwent normal developmental sequences. Under these conditions, vital staining with the Bolton-Hunter reagent disproportionately labeled exterior proteins, and it seems likely that the Bolton-Hunter reagent is an especially good vital stain for cell surface and cell membrane proteins. The Bolton-Hunter stain is covalently bound, is not reutilizable, and appears not to disrupt natural physiological and developmental processes. Thus, we used the Bolton-Hunter reagent to follow the natural life spans of proteins in vivo and we were able to distinguish particularly long-lived proteins in Xenopus embryos.     

An improved silver stain for developing nervous tissue.

A reduced silver technique using physical development to stain embryonic nervous tissue is described. Brains are fixed in Bodian's fixative. Paraffin sections are pretreated with 1% chromic acid or 5% formol. They are impregnated with 0.01% silver nitrate dissolved in 0.1 M boric acid/sodium tetraborate buffer of pH 8 or with silver proteinate. Finally they are developed in a special physical developer which contains 0.1% silver nitrate, 0.01-0.1% formol as reducing agent, 2.5% sodium carbonate to buffer the solution at pH 10.3, 0.1% ammonium nitrate to prevent precipitation of silver hydroxide, and 5% tungstosilicic acid as a protective colloid. The development takes several minutes in this solution, thus the intensity of staining can be controlled easily. The method yields uniform, complete and reproducible staining of axons at all developmental stages of the nervous tissue and is easy to handle.     

Fetuin‐A in the developing brain

The serum protein fetuin‐A is essential for mineral homeostasis and shows immunomodulatory functions, for example by binding to TGF superfamily proteins. It proved neuroprotective in a rat stroke model and reduced lethality after systemic lipopolysaccharide challenge in mice. Serum fetuin‐A concentrations are highest during intrauterine life. Different species show intrauterine cerebral fetuin‐A immunoreactivity, suggesting a contribution to brain development. We therefore aimed at specifying fetuin‐A immunoreactivity in brains of newborn rats (age P0–P28) and human neonates (20–40 weeks of gestation). In humans and rats, fetuin‐A was found in cortex, white matter, subplate, hippocampus, subventricular zone, and ependymal cells which supports a global role for brain function. In rats, overall fetuin‐A immunoreactivity decreased with age. At P0 fetuin‐A immunoreactivity affected most brain structures. Thereafter, it became increasingly restricted to distinct cells of the hippocampus, cingular gyrus, periventricular stem cell layer, and ependyma. In ependymal cells the staining pattern complied with active transependymal transport from cerebrospinal fluid. Double immunofluorescence studies revealed colocalization with NeuN (mature neurons), beta III tubulin (immature neurons), GFAP (astrocytes), and CD68 (activated microglia). This points to a role of fetuin‐A in different brain functional systems. In human neonatal autopsy cases, frequently affected from severe neurological and non‐neurological diseases, fetuin‐A immunoreactivity was heterogeneous and much less associated with age than in healthy tissues studied earlier, suggesting an impact of exogeneous noxious factors on fetuin‐A regulation. Further research on the role of fetuin‐A in the neonatal brain during physiological and pathological conditions is recommended. © 2012 Wiley Periodicals, Inc. Develop Neurobiol 73: 354–369, 2013