The Use of Agents Which Improve Staining of Fresh Human Fibrocartilage

Comparisons of three agents were undertaken to improve the bulk staining of fresh human fibrocartilage with gold chloride for neural elements. The medial meniscus of the knee was the experimental tissue. Dimethyl sulfoxide (DMSO) increased the penetration of the stain but had a negative effect on the deposition of the gold on the tissue site. Sodium borohydride apparently reacted with tissue aldehydes decreasing the background enough to give the impression of an improved staining reaction. Triton X-100, the agent of choice, solubilized the collagen protein of the menisci sufficiently to allow penetration of the stain and, being nonionic, did not react with the gold chloride solution.     

Improved gold chloride staining method for anatomical analysis of sensory nerve endings in the shoulder capsule and labrum as examples of loose and dense fibrous tissues

Consistency in gold chloride staining is essential for anatomical analysis of sensory nerve endings. The gold chloride stain for this purpose has been modified by many investigators, but often yields inconsistent staining, which makes it difficult to differentiate structures and to determine nerve ending distribution in large tissue samples. We introduce additional steps and major changes to the modified Gairns’ protocol. We controlled the temperature and mixing rate during tissue staining to achieve consistent staining and complete solution penetration. We subjected samples to sucrose dehydration to improve cutting efficiency. We then exposed samples to a solution containing lemon juice, formic acid and paraformaldehyde to produce optimal tissue transparency with minimal tissue deformity. We extended the time for gold chloride impregnation 1.5 fold. Gold chloride was reduced in the labrum using 25% formic acid in water for 18 h and in the capsule using 25% formic acid in citrate phosphate buffer for 2 h. Citrate binds gold nanoparticles, which minimizes aggregation in the tissue. We stored samples in fresh ultrapure water at 4° C to slow reduction and to maintain color contrast in the tissue. Tissue samples were embedded in Tissue Tek and sectioned at 80 and 100 μm instead of using glycerin and teasing the tissue apart as in Gairns’ modified gold chloride method. We attached sections directly to gelatin subbed slides after sectioning with a cryostat. The slides then were processed and coverslipped with Permount. Staining consistency was demonstrated throughout the tissue sections and neural structures were clearly identifiable.     

Improved Gold Chloride Procedure for Nerve Staining in Whole Mounts of Rat Corneas

The purpose of this study was to modify the gold chloride procedure for studies of total innervation in corneal whole mounts to provide a decrease in nonspecific background staining and to eliminate the progressively deteriorating stain quality of standard gold chloride techniques. Modifications included use of cryo-protective agents, mechanical removal of Descemet's membrane-endothelium complex prior to fixation, treatment with alpha amylase, and halting the reduction of gold chloride to metallic gold using Kodak rapid fixer with hardener. Rat corneas were stored at-70 C in O. C.T. compound. The Descemet's membrane-endothelium complex was removed after thawing, and corneas were fixed in 4% NaPO₄-buffered paraformaldehyde with 8% sucrose. Fixed corneas were incubated in NaPO₄-buffered saline containing alpha amylase, placed in 100% lemon juice, then in 1% gold chloride solution, transferred to glacial acidic acid, placed in rapid fixer, rinsed in NaPO₄-buffered saline, and dehydrated in graded alcohols. Flat mounts of whole corneas were examined using contralateral corneas as controls. Freezing corneas in O. C.T. compound, removal of the Descemet's membrane-endothelium complex, and treatment with alpha amylase reduced nonspecific background staining compared to controls. Treatment with Kodak rapid fixer prevented the deterioration of staining quality for at least 8 months. These improvements allow the gold chloride technique to be used with immunohisto-chemical procedures where the reaction products would be obscured by background staining.     

A modified gold chloride technique for optimal impregnation of nerves within corneal whole mounts and dura of the albino rat

Ranvier's method of staining tissue whole mounts with gold chloride to visualize nerve fibers was modified by lengthening the incubation time in gold chloride and reducing the time in acidulated water. These simple modifications of an old technique give consistent impregnation of nerve fibers with light background staining in whole mounts of cornea and dura.     

Investigation of immunogold-silver staining by electron microscopy

Deposition of metallic silver on colloidal gold immunoreagents has been shown to be a very sensitive immunostaining technique capable of detecting low levels of immunoreactivity in tissue sections. Using electron microscopy we have shown that immunolabelling is highest with small sizes of gold which can penetrate sections better and achieve higher densities of particles in the section than larger particles. Chemical permeabilisation of the embedding medium aids the penetration of colloidal gold. The silver enhancement step in immunogold-silver staining was shown to be progressive, allowing optimisation of staining and the selection of the final size of silver deposits required. Some poorly understood features of the technique are rationalised and the additional knowledge gained will aid the wider application of this method.     

Investigation of immunogold-silver staining by electron microscopy

Summary Deposition of metallic silver on colloidal gold immunoreagents has been shown to be a very sensitive immunostaining technique capable of detecting low levels of immunoreactivity in tissue sections. Using electron microscopy we have shown that immunolabelling is highest with small sizes of gold which can penetrate sections better and achieve higher densities of particles in the section than larger particles. Chemical permeabilisation of the embedding medium aids the penetration of colloidal gold. The silver enhancement step in immunogold-silver staining was shown to be progressive, allowing optimisation of staining and the selection of the final size of silver deposits required. Some poorly understood features of the technique are rationalised and the additional knowledge gained will aid the wider application of this method.     

The Surface Staining of Embedded Tissues

The staining procedure is based on the theory that the freshly cut surface of embedded material will absorb stain only in the exposed tissue elements, provided that the embedding compound itself will not absorb the staining fluid. Concentrated stains are used for short intervals to insure minimum penetration. For paraffin embedded materials: (1) Cut block, preferably on microtome, to the desired tissue surface. (2) Rinse in absolute alcohol. (3) Float face down in stain. (Ripe, concentrated alum hematoxylin—Galigher's formula recommended—will stain in 10 to IS minutes. Heidenhain's iron hematoxylin works exceptionally well in some cases.) Mordant 20% alum 5 to 10 minutes, briefly rinse, and stain comparable 5 to 10 minutes in 1 to 1.5% hematoxylin. (4) Allow to become blue in tap water (for hematoxylin stains). (5) Counter-stain if desired. (6) Dehydrate in absolute alcohol for not more than 10 minutes. (7) Dry for 15 to 20 minutes. (8) Trim block to 2-3 mm. and mount between two cover glasses by use of microflame. Attach mount to slide with balsam. For celloidin embedded materials: (1) Dehydrate block with 90% alcohol, phenol-toluene, finally pure toluene. (2) Rinse cut surface with 90% alcohol, then apply stain. (3) Wash, after hematoxylin stains, counterstain if desired. (4) Dehydrate surface, 90% alcohol, phenol toluene, pure toluene, and mount in medium dissolved in toluene.

Possible applications of surface staining technic are suggested and illustrated.     

A luminescent ruthenium complex for ultrasensitive detection of proteins immobilized on membrane supports

SYPRO Ruby protein blot stain provides a sensitive, gentle, fluorescence-based method for detecting proteins on nitrocellulose or polyvinylidene difluoride (PVDF) membranes. SYPRO Ruby dye is a permanent stain composed of ruthenium as part of an organic complex that interacts noncovalently with proteins. Stained proteins can be excited by ultraviolet light of about 302 nm or with visible light of about 470 nm. Fluorescence emission of the dye is approximately 618 nm. The stain can be visualized using a wide range of excitation sources utilized in image analysis systems including a UV-B transilluminator, 488-nm argon-ion laser, 532-nm yttrium-aluminum-garnet (YAG) laser, blue fluorescent light bulb, or blue light-emitting diode (LED). The detection sensitivity of SYPRO Ruby protein blot stain (0.25-1 ng protein/mm(2)) is superior to that of amido black, Coomassie blue, and india ink staining and nearly matches colloidal gold staining. SYPRO Ruby protein blot stain visualizes proteins more rapidly than colloidal gold stain and the linear dynamic range is more extensive. Unlike colloidal gold stain, SYPRO Ruby protein blot stain is fully compatible with subsequent biochemical applications including colorimetric and chemiluminescent immunoblotting, Edman-based sequencing and mass spectrometry.     

Gold enhancement of gold-labeled probes: gold-intensified staining technique (GIST)

In this report we present a staining method in which gold chloride is used to enhance the size of gold colloids. We show the utility of this technique when used in conjunction with small gold colloids, i.e., 5 nm, 4 nm, and 2.6 nm. Post-embedding staining of epoxy-embedded, gold-labeled mouse LM fibroblasts showed that staining with 0.1% gold chloride facilitated the visualization of the smallest gold colloids.     

A simple method using alizarin red S for the detection of calcium in epoxy resin embedded tissue

This report presents a simple procedure for staining 1-2 microns epoxy plastic sections of cells and mineralizing matrix present in fetal bovine bone tissue cultures. A 0.3% aqueous toluidine blue O solution was used as a cellular stain and was followed with 2% alizarin red S for the detection of calcium at sites of mineralization. Effects of concentration and pH of alizarin red S on the penetration of epon embedded thick sections were investigated. Optimal staining was achieved with a 2% aqueous alizarin red S solution adjusted to a pH of 5.5-6.5. This staining procedure provides unusually clear contrast between mineral and bone cells in plastic sections for light microscopy.     

A Simple Method Using Alizarin Red S For the Detection of Calcium in Epoxy Resin Embedded Tissue

This report presents a simple procedure for staining 1-2 μm epoxy plastic sections of cells and mineralizing matrix present in fetal bovine bone tissue cultures. A 0.3% aqueous toluidine blue 0 solution was used as a cellular stain and was followed with 2% alizarin red S for the detection of calcium at sites of mineralition. Effects of concentration and pH of alizarin red S on the penetration of epon embedded thick sections were investigated Optimal staining was achieved with a 2% aqueous alizarin red S solution adjusted to a pH of 5.5-6.5. This staining procedure provides unusually clear contrast between mineral and bone cells in plastic sections for light microscopy.     

Bodian's silver impregnation of endocrine cells. A tentative explanation to the staining mechanism

We have recently shown that a variety of proteins, including albumin and immunoglobulins conjugated to colloidal gold, strongly binds to certain basic peptide sequences, and neurohormonal peptides. Silver proteinate, used in the classical Bodian's neurohistological procedure, is now shown to bind to the same peptide sequences in cytochemical model systems. In tissue, gastrin cells and glucagon cells have been reported to show strong unspecific immunocytochemical staining and these cell types also stain in the Bodian's procedure. These results suggest that certain types of unspecific immunocytochemical staining and the Bodian's silver staining method may depend upon a common mechanism, involving binding of labelled or aggregated protein to basic and hydrophobic sequences in tissue.     

Direct Staining of Reticular Fibers with Gold Chloride

Reticular fibers are selectively stained in paraffin sections of formalin-fixed or Bouin's-fixed tissue as follows: 1% aqueous solution of gold chloride for 20 min, followed by a 10 min immersion in an aqueous solution containing 5% Na₂CO₃ and 0.5% KOH. The sections then are placed in a 5% aqueous solution of KI for 2 min. Counterstaining with a 0.25% aqueous solution of methylene blue chloride is optional. The reticular fibers stain dark pink; the collagen bundles are a light pink to straw color without the counterstain, or a light blue color when the methylene blue is used.     

Suppression of Costaining of Nonnervous Tissue in Protargol Technics

If, in the procedure of staining nerve fibers in mounted paraffin sections with Protargol according to Bodian, the reduction after toning with gold chloride is executed in a solution of 3-6 drops of aniline oil in 100 ml of 50% alcohol instead of in the prescribed oxalic acid solution, the selectivity of the staining of peripheral nerves is increased. This is effected by a reduction in the intensity of the staining of nonnervous tissue elements. However, at the same time the staining of nonnervous tissue is richer in details and consequently more satisfactory from a histological point of view than it is according to the original method of Bodian or the modification of this method by Ziesmer (1951).     

Palladium Toning of Silver-Impregnated Reticular Fibers

Palladium toning is much less expensive than gold toning. Ten minutes in 0.05% potassium hexachloropalladate in 4 N hydrochloric add tones silver-impregnated reticular fibers as well as 3 min in 0.2% aqueous gold chloride does. Differences in toning of the background depend on the silver stain.     

Palladium toning of silver-impregnated reticular fibers

Palladium toning is much less expensive than gold toning. Ten minutes in 0.05% potassium hexachloropalladate in 4 N hydrochloric acid tones silver-impregnated reticular fibers as well as 3 min in 0.2% aqueous gold chloride does. Differences in toning of the background depend on the silver stain.     

SDS聚丙烯酰胺凝胶电泳快速染色新方法的研究

通过几种金融盐溶液对SDS聚丙烯酰胺凝胶电泳染色的实验表明,0.25mol/L的CaCl2和MgCl2溶液能够对蛋白质进行有效的染色,经这2种溶液染色的蛋白质都能够从凝胶中洗脱回收。尤其是CaCl2法灵敏度更高,而且蛋白质条带形成之后也十分稳定,所以在运用制备电泳纯化蛋白质时这种新的染色方法较适用。     

Colloidal gold probes in immunocytochemistry. An optimization of their application in light microscopy by use of silver intensification procedures

Colloidal gold particles are the markers of choice for ultrastructural localization of antigens. By reducing gold chloride with tannic acid and trisodium citrate, a broad range of narrowly determined particle sizes can be obtained. Such particles can easily be coupled to a number of proteins and the resulting conjugates are conveniently purified on a gel-chromatography column. Their application in light microscopy requires an amplification step with a silver physical developer. Silver-intensified colloidal gold probes can advantageously be used for immunostaining of cryostat, paraffin and plastic sections. Moreover, permeabilized cultured cells and whole-mount preparations can also be stained with gold-silver techniques. Silver intensification does not affect reactivity of a number of tissue antigens, thus permitting double staining combinations with immunoperoxidase or immunofluorescence methods.     

Modified Elastic Tissue-Masson Trichrome Stain

A combined elastic tissue-Massou technique is presented which stains elastic fibers of all sizes, nuclei and connective tissue. The modified elastic tissue stain consists of hematoxylin, ferric chloride and Verhoeffs iodine; nuclei and elastic fibers are stained blue-black in six minutes without differentiation. By contrast, cytoplasmic elements are stained red, (Biebrich scarlet-acid fuchsin) and collagen is stained green (light green) or blue (aniline blue). The entire staining procedure takes approximately one hour.     

Papanicolaou stain: Is it economical to switch to rapid, economical, acetic acid, papanicolaou stain?

OBJECTIVE: To standardize an inexpensive and rapid Papanicolaou staining technique with limited ethanol usage. STUDY DESIGN: Smears from 200 patients were collected (2 per patient) and fixed in methanol. Half were subjected to conventional Papanicolaou and half to stain ing with rapid, economical, acetic acid Papanicolaou (REAP) stain. In REAP, pre-OG6 and post-OG6 and post-EA36 ethanol baths were replaced by 1% acetic acid and Scott's tap water with tap water. Hematoxylin was preheated to 60 degrees C. Final dehydration was with methanol. REAP smears were compared with Papanicolaou smears for optimal cytoplasmic and nuclear staining, stain preservation, cost and turnaround time. RESULTS: With the REAP method, cytoplasmic and nuclear staining was optimal in 181 and 192 cases, respectively. The staining time was considerably reduced, to 3 minutes, and the cost per smear was reduced to one fourth. The staining quality remained good in all the smears for > 2 years. CONCLUSION: REAP is a rapid, cost-effective alternative to Papanicolaou stain. Though low stain penetration in large cell clusters is a limitation, final interpretation was not compromised.