An improved colloidal iron stain for glomerular sialoproteins

The thick sialoglycoprotein coat of the glomerular epithelial cells is a part of the glomerular filtration barrier. It can be demonstrated in paraffin histoslogy by means of the colloidal iron procedure. We have studied the special requirements of tissue processing to obtain reproducible results by this staining method. The requirements include proper fixation, avoiding heat during sectioning and ample rehydration. We have further tested a colloidal iron-PAS procedure for simultaneous demonstration of the glomerular epithelial coat and the basement membrane.     

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.     

An improved formulation of the zirconyl hematoxylin stain for acidic mucins

Zirconyl hematoxylin stains acidic mucins darkly and specifically using a solution of 100 mg hematoxylin, 5 ml ethanol, 5 ml 0.5% sodium iodate, 400 mg zirconyl chloride octahydrate, and 30 ml 25% aqueous glycerol. The stain is especially advantageous for studying goblet cells and Paget cells.     

An improved formulation of Nocht's azure-eosin stain for plastic embedded material

The popular azure-eosin stain of Nocht has been reformulated to provide consistent, intense staining of semithin sections of water-miscible methacrylate embedded tissues.     

An improved formulation of the zirconyl hematoxylin stain for acidic mucins

Zirconyl hematoxylin stains acidic mucins darkly and specifically using a solution of 100 mg hematoxylin, 5 ml ethanol, 5 ml 0.5% sodium iodate, 400 mg zirconyl chloride octahydrate, and 30 ml 25% aqueous glycerol. The stain is especially advantageous for studying goblet cells and Paget cells.     

An interesting case of colloidal iron positivity

Summary An interesting case of a colloidal iron (CI) positive basophilic substance in the adrenal medullary cells of amphibia and reptilia is reported here. The substance, however, does not stain by alcian blue (AB). It is negative to PAS, Azure A, aldehyde fuchsin, AB at pH 1 and MgCl₂ — AB though orthochromatically stained by toluidine blue at pH 3. More work is needed to establish the exact nature of the CI positive material.     

An improved formulation of the zirconyl hematoxylin stain for acidic mucins.

Zirconyl hematoxylin stains acidic mucins darkly and specifically using a solution of 100 mg hematoxylin, 5 ml ethanol, 5 ml 0.5% sodium iodate, 400 mg zirconyl chloride octahydrate, and 30 ml 25% aqueous glycerol. The stain is especially advantageous for studying goblet cells and Paget cells.     

一种改进的丛枝菌根染色方法

研究改进了Vierheilig等描述的AM菌根染色法:将根样于20%KOH溶液中60℃水浴透明40-120 min,5%醋酸酸化5min后,用5%醋酸墨水染色液(派克纯黑书写墨水Quink),于60℃水浴染色30 min,清水浸泡脱色(14h)后即可镜检。根皮层细胞内AM真菌的丛枝结构清晰可见,并且能够明确地分辨AM真菌与其它未知真菌。此外,Quink初染后,再经过SudanⅣ复染(60℃、60 min),70%乙醇脱色5min,暗隔真菌的透明菌丝内所积聚的脂类颗粒被SudanⅣ染上鲜红色,在复式显微镜下能够观察到此类透明菌丝在根皮层组织内的存在状况。采用甘油明胶为封固剂制片,根的染色效果可以保存长久。此项技术可以对同一种植物的多个根样进行同步的透明和染色处理,而且操作简便、低毒性、成本低廉、染色效果极佳,适用于野生和栽培草本植物AM菌根的染色和制片观察。     

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.     

An evaluation of colloidal solutions for normothermic perfusion of rabbit hearts: An improved perfusate containing Haemaccel

Rabbit hearts were perfused at 37 °C with the aim of establishing a relatively simple and preferably synthetic perfusate which would give reliable and consistent perfusion performances for periods of several hours. Of the perfusates devised and tested, a 1.75% solution of Haemaccel most fully satisfied the above criteria and maintained myocardial function for 30.0 ± 4.0 hr.     

An activity stain for dihydroxy-acid dehydratase

An activity stain has been devised for the dihydroxy-acid dehydratase. When applied to polyacrylamide gel electropherograms of crude soluble extracts of Escherichia coli, it detected a single electromorph. The intensity of staining increased with the amount of extract protein applied to the gel. Activity staining demonstrated that (a) anaerobically grown cells contain more extractable dehydratase activity than do aerobically grown cells; (b) exposure of E. coli to 4.2 atm O2 caused virtually complete loss of activity; (c) exposure of cells to paraquat or plumbagin in the presence of dioxygen, but not in its absence, caused a massive loss of activity. These data illustrate the utility of this activity stain and demonstrate that the dehydratase is inactivated by O2- generated within cells.     

An improved combined cholinesterase stain and silver impregnation method for quantitative analysis of innervation patterns in frog muscle

The original method combining Karnovsky's cholinesterase stain and Bodian silver impregnation has been modified to stain both myelinated and unmyelinated axons and to reduce background staining. The improvements were obtained by adding nitric acid to a paraformaldehyde-acetone fixative and by carrying out the silver impregnation of axons in an alcoholic solution. The method is especially suitable for quantitative estimation of the different kinds of nerve sprouting as well as for study of the remodeling of neuromuscular junctions in normal and experimental frog muscles.     

A simple improved method to stain Thiobacillus.

Best stains are obtained by immersion of the fixed smear in 1.5-2% acid fuchsin at pH 3.2-3.6.     

A stain for iron-containing proteins sensitive to nanogram levels of iron

A method for staining iron proteins on polyacrylamide gel electropherograms is described. It is based upon catalysis, by iron, of the oxidation of diaminobenzoic acid by hydrogen peroxide. Bands containing as little as 5 ng of protein-bound iron can be visualized by this method.     

An improved cell recovery method for iron oxidizing bacterial (IOB) enrichments

Two cell recovery methods for IOB enrichments were evaluated for DNA extraction and further PCR-based 16S rRNA gene clone library creation. One was a published method consisting of heating plus oxalic acid treatment and the other one was a new method based on enzymatic agarose digestion (using beta-agarase I). The results indicated that the enzymatic method was much gentler on IOB cells and yielded an approximately 5000-fold higher DNA mass than the published method. The 16S rRNA gene clone library developed from the beta-agarase I treated IOB enrichments indicated a high IOB community diversity with sequences in alpha-, beta-, gamma-, delta-, epsilon-Proteobacteria, unclassified Proteobacteria, unclassified Bacteroidetes and unclassified Bacteria. In contrast, the published method resulted in mainly gamma-Proteobacterial clone sequences. In addition, only the cells recovered by agarase treatment were amenable to direct fluorescence in situ hybridization (FISH). Therefore, we propose that the agarase method is a better IOB cell recovery method, because it is simpler, faster, and retains more genetic diversity.     

A combined Bodian-Nissl stain for improved network analysis in neuronal cell culture

Bodian and Nissl procedures were combined to stain dissociated mouse spinal cord cells cultured on coverslips. The Bodian technique stains fine neuronal processes in great detail as well as an intracellular fibrillar network concentrated around the nucleus and in proximal neurites. The Nissl stain clearly delimits neuronal cytoplasm in somata and in large dendrites. A combination of these techniques allows the simultaneous depiction of neuronal perikarya and all afferent and efferent processes. Costaining with little background staining by either procedure suggests high specificity for neurons. This procedure could be exploited for routine network analysis of cultured neurons.