ABSTRACT
Highly reproducible results in molecular biology depend a lot on effective staining and destaining methods. Silver staining of polyacrylamide DNA and protein gel has been adopted widely in the molecular biology laboratories for detecting a very low nanogram range of sample. An efficient staining of a polyacrylamide gel requires a number of well controlled and highly sensitive steps that often becomes tiresome when done manually or when there are a number of gels to be stained simultaneously. Since, silver staining is a multistep procedure that requires proper fixation and exchange of substance, a reliable protocol is necessary and a simple apparatus may be an added advantage to carry out the steps with ease and safety. Here, we describe a simple and cost effective device made from off-the-shelf components for some established silver staining protocols. Staining is done on a tray while six graduated bottles with a liquid delivery stopcock each, is connected to the tray through silicon tubing. The used up solution is drained off completely from the staining tray through a liquid outlet stopcock using vacuum pressure. The system is fixed with a camera connected to a computer for effective control of the staining process in each step. The apparatus provides the researchers with efficient staining and real time monitoring of gels without the need for handling toxic chemicals.
Subject(s)
Silver Staining/instrumentation , Acrylic Resins/chemistry , DNA/chemistry , Electrophoresis, Polyacrylamide Gel/economics , Electrophoresis, Polyacrylamide Gel/methods , Electrophoresis, Polyacrylamide Gel/standards , Indicators and Reagents , Proteins/chemistry , Reference Standards , Silver Staining/economics , Silver Staining/methodsABSTRACT
Silver staining is widely used to detect protein in polyacrylamide gels when high sensitivity is required. A simple and rapid protocol for silver staining of proteins following PAGE was developed in the present study. The number of steps was reduced compared to conventional protocol by combining fixing, rinsing, and soaking into a single impregnating step, thus achieving detection of proteins in 20 min. The present method is as sensitive as current protocols with the advantage of saving time and costs.
Subject(s)
Electrophoresis, Polyacrylamide Gel/methods , Proteins/chemistry , Silver Staining/methods , Electrophoresis, Polyacrylamide Gel/economics , Sensitivity and Specificity , Silver Staining/economics , Time FactorsABSTRACT
Onychomycosis is a frequently treated fungal infection of the nail plate with morbidity in high-risk populations. The diagnosis often relies on histopathologic analysis of nail plate specimens with the assistance of special stains. Pathologists utilize periodic acid schiff (PAS) and/or Gomori methenamine silver (GMS) stains to highlight fungi within the nail plate. In a recent study of 51 PAS-negative nail cases, it was concluded that GMS is superior to PAS in the diagnosis of onychomycosis. We expand on this study by investigating a larger number of PAS-negative nail clippings determining whether GMS or PAS is superior in highlighting fungi in additional sections. There was no difference in the sensitivity of PAS vs. GMS (4.2 vs. 4.3%, p = 0.57); however, PAS was found to be significantly less expensive by 2.6-fold. Taken together, these data suggest that the PAS stain is the optimal method for diagnosing onychomycosis.
Subject(s)
Foot Dermatoses/diagnosis , Hand Dermatoses/diagnosis , Onychomycosis/diagnosis , Periodic Acid-Schiff Reaction , Humans , Periodic Acid-Schiff Reaction/economics , Sensitivity and Specificity , Silver Staining/economicsABSTRACT
A convenient silver staining method for protein in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels is described. The method is previsible, sensitive, and mass spectrometry (MS) compatible. Two visible counter ion dyes, ethyl violet (EV) and zincon (ZC), were used in the first staining solution with a detection limit of 2 to 8 ng/band in approximately 1h. The dye-stained gel can be further stained by silver staining, which is based on acidic silver staining employing ZC with sodium thiosulfate as silver ion sensitizers. Especially, ZC has silver ion reducing power by cleavage of the diazo bond of the dye during silver reduction. The second silver staining can be completed in approximately 1h with a detection limit of 0.2 ng/band.
Subject(s)
Analytic Sample Preparation Methods/methods , Electrophoresis, Polyacrylamide Gel/methods , Proteins/analysis , Silver Staining/methods , Analytic Sample Preparation Methods/economics , Animals , Cattle , Coloring Agents/chemistry , Costs and Cost Analysis , Linear Models , Mass Spectrometry , Proteins/chemistry , Proteomics , Rosaniline Dyes/chemistry , Sensitivity and Specificity , Silver Staining/economics , Time Factors , Zinc/chemistryABSTRACT
OBJECTIVE: To develop a cost-effective, reliable and safe method of providing fungal control slides for routine use in pathology laboratories. STUDY DESIGN: A set of easily available, low-cost material was tested to obtain fungal colonies on substrate adequate for paraffin-embedded sections or smears. RESULTS: Such material as cheese is a simple, inexpensive and practical culture medium for silver-positive fungi. A batch of paraffin blocks can be prepared to maintain a stock of control material in the laboratory. CONCLUSION: It is useful to maintain fungal colonies to produce staining control specimens using small pieces of refrigerated cheese to easily produce silver-staining control specimens or smears embedded in paraffin, reducing the risk of accidental exposure to potentially infective pathogens in the laboratory. This method might also be a good alternative for conserving routine surgical specimens, considering the currently decreasing numbers of necropsy and large specimens, particularly from immunosuppressed and infected patients.
