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.     

A combined silver and acetylcholinesterase method for staining intramuscular innervation.

A combined acetylcholinesterase and silver stain for demonstrating the intramuscular innervation of fresh frozen tissue is described. Intramuscular nerves, subterminal axons, and motor end plates are simultaneously stained brown or black with minimal staining of connective tissue and muscle fibers in longitudinal sections 30-100 mu thick. The method has been applied to fetal and adult rat, porcine, and bovine skeletal muscle. Antemortem and postmortem tissue samples stained equally well. The method facilitates simultaneous appreciation of morphological alterations in nervous and muscular tissues; in clinical and research laboratories alike it is of value when muscle abnormalities which may be related to disorders of nervous origin are studied. Compared with other published procedures this method has shorter time requirements, uses fresh frozen tissue, and displays superior staining characteristics.     

An improved method for cytogenetic analysis of meiotic aneuploidy in rodent and frog spermatocytes

Methods are described for the attachment of isolated spermatocytes to glass slides and the subsequent hypotonic swelling and gradual fixation of the metaphase I and metaphase II cells. The methods minimize cell loss and cell disruption and meiotic metaphase chromosomes become spread within residual cytoplasm thus reducing artefactual chromosome loss. Metaphase II complements from mouse, rat and frog spermatocytes prepared by these procedures had relatively low frequencies of hypoploidy (0.5-1.6%). Bivalent loss was not detected in 916 metaphase I complements. Injection of 0.1 mg/kg demecolcine into mice increased the incidence of metaphase II hypoploidy 8-fold. The hypoploid and hyperploid frequencies here increased equally. The results suggest that the methods described may be useful for the analysis of mechanisms of meiotic aneuploidy including aneuploidy resulting from chromosome loss during meiosis I.     

An improved method for quantitative analysis of total fructans in plant tissues

Current methods for measuring fructan levels in plant tissues are time-consuming and costly. They often involve multiple or sequential extractions, enzymatic or acid hydrolysis of fructan polymers, and multiple HPLC runs to quantify fructan-derived hexoses. Here we describe a new method that requires a single extraction step, followed by selective precipitation of fructans by acetone, acid hydrolysis of the precipitate, and a short (10 min) HPLC run to complete the procedure. We used perennial ryegrass samples to show that the new method has similar sensitivity, but better reproducibility, than a more complex method that is widely used. We have used the new method to study developmentally related changes in fructan levels in glasshouse-grown perennial ryegrass plants.     

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.     

A method for silver impregnation of mammary myoepithelia

Histochemical methods for microscopic visualization of mammary myoepithelial cells all yielded considerable variation in completeness of myoepithelial cell staining. Although extremely variable, silver impregnation occasionally gave tissue sections containing myoepithelia having excellent microanatomical detail and contrast with other tissue elements. Consequently, sources of variation in the silver technique were considered. Composition of the tissue fixative and pH of the silver impregnating solution were most critical. A final method is presented which gives consistent, complete silver impregnation of myoepithelia, where both the cell body and cell processes are clearly evident. The staining procedure is not light sensitive, nor is acid cleaning of glassware necessary. Tissue sections from lactating mouse, rat, hamster and goat are presented; tissue from other species should stain as well. The procedure should greatly facilitate the study of the function of myoepithelial cells and the visualization of these cells in mammary pathology.     

Phosphoinositides in frog skeletal muscle: a quantitative analysis

The contents of major phospholipids per g of wet wt. in frog skeletal muscle are: 5.3 mumol PC; 1.4 mumol PE; 1 mumol SM; 0.4 mumol PtdIns; 0.3 mumol CL; and 0.13 mumol PS. The quantities of polyphosphoinositides per g of wet wt. are: 181 nmol PtInsP; 28 nmol PtdInsP2; and 8 nmol lyso-PtdInsP2. The specific activity of labelling of the total muscle ATP attained by external incubation with [32P]Pi was found to be 57 dpm/nmol x g muscle wet wt. PtdInsP2, the highest labelled polyphosphoinositide, showed a specific activity of 64,000 dpm/nmol per g muscle wet wt., suggesting that high specific activity ATP may be compartmentalized in the local environment of the triads and used as a substrate by the PtdIns and PtInsP kinase in that region. PtdInsP2 which is the immediate precursor for the release of InsP3, is found at a significant concentration and strategically located for its postulated role as a substrate for the action of phosphoinositidase C. The presence of a novel endogenous polyphosphoinositide, lyso-PtdInsP2, in animal tissues is reported for the first time. Electrical stimulation leads towards a rapid catabolization of polyphosphoinositides revealed by reductions in the 3H- and 32P-labelling, suggesting that muscle excitation is associated with the activation of breaking down of polyphosphoinositides.     

A modified PAS stain combined with immunofluorescence for quantitative analyses of glycogen in muscle sections

Simultaneous analyses of glycogen in sections with other subcellular constituents within the same section will provide detailed information on glycogen deposition and the processes involved. To date, staining protocols for quantitative glycogen analyses together with immunofluorescence in the same section are lacking. We aimed to: (1) optimise PAS staining for combination with immunofluorescence, (2) perform quantitative glycogen analyses in tissue sections, (3) evaluate the effect of section thickness on PAS-derived data and (4) examine if semiquantitative glycogen data were convertible to genuine glycogen values. Conventional PAS was successfully modified for combined use with immunofluorescence. Transmitted light microscopic examination of glycogen was successfully followed by semiquantification of glycogen using microdensitometry. Semiquantitative data correlated perfectly with glycogen content measured biochemically in the same sample (r²=0.993, P<0.001). Using a calibration curve (r²=0.945, P<0.001) derived from a custom-made external standard with incremental glycogen content, we converted the semiquantitative data to genuine glycogen values. The converted semiquantitative data were comparable with the glycogen values assessed biochemically (P=0.786). In addition we showed that for valid comparison of glycogen content between sections, thickness should remain constant. In conclusion, the novel protocol permits the combined use of PAS with immunofluorescence and shows valid conversion of data obtained by microdensitometry to genuine glycogen data.     

A combined silver and cholinesterase method for studying exact relations between the pre- and the postsynaptic elements at the frog neuromuscular junction.

Combination of Karnovsky's cholinesterase staining with silver impregnation of axons (modified Bodian's technique) offers a new means of studying the relation between the pre- and postsynaptic elements in the frog neuromuscular junction. The method can be applied to whole muscles so that synapses of individual superficial muscle fibers which have previously been investigated by electrophysiological techniques can be identified after staining. In this way synaptic activity can be correlated with such synaptic features as number of axon branches, length of the occupied synaptic gutter, axonal sprouts, etc. The distinction between occupied and unoccupied parts of the synaptic gutters is useful when studying reinnervation, regression, or growth of a synapse.     

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。     

Comparative analysis of an improved thioflavin-s stain, Gallyas silver stain, and immunohistochemistry for neurofibrillary tangle demonstration on the same sections.

An improved thioflavin-S stain, Gallyas silver stain, and two immunostainings were quantitatively compared for demonstration of neurofibrillary tangles (NFTs) on the same sections. Sections of hippocampal formation from seven cases of Alzheimer's disease (AD) were immunofluorescently stained with a commercially available polyclonal NFT antibody or a PHF-1 monoclonal antibody, followed by an improved thioflavin-S stain, and finally by Gallyas silver staining. The thioflavin-S method was improved by using a combination quenching method that removes background autofluorescence without remarkable tissue damage and by post-treatment with concentrated phosphate buffer, which minimizes photobleaching. PHF-1 or NFT immunostaining is much less sensitive than the improved thioflavin-S staining and Gallyas silver staining, particularly in the transentorhinal region. Moreover PHF-1 immunoreactivity varied greatly among AD individuals. Thioflavin-S staining and Gallyas silver staining show almost the same sensitivity in NFT demonstration, but only the former depends on the secondary protein structure of NFTs. This study suggests that the improved thioflavin-S staining is a simple, sensitive, and consistent method for demonstration of neurofibrillary pathology.     

Detection of protein in polyacrylamide gels using an improved silver stain

A much improved silver staining procedure for the detection of protein in polyacrylamide gels of 0.8-3.0 mm thickness is described. It achieves very high sensitivity (detecting less than 0.01 ng bovine serum albumin/mm2) by overstaining and subsequently removing nonspecific background stain using a modified, reliable destaining procedure. Maximum sensitivity follows prediamine equilibration in 0.1% (w/v) formaldehyde solution. With two-dimensional electrophoresis the improved staining procedure reveals greater than 200 polypeptides in unconcentrated human urine and greater than 150 polypeptides in a single human fingerprint.