Picrosirius staining plus polarization microscopy, a specific method for collagen detection in tissue sections

Synopsis Sirius Red, a strong anionic dye, stains collagen by reacting, via its sulphonic acid groups, with basic groups present in the collagen molecule. The elongated dye molecules are attached to the collagen fibre in such a way that their long axes are parallel. This parallel relationship between dye and collagen results in an enhanced birefringency.Examination of tissue sections from 15 species of vertebrates suggests that staining with Sirius Red, when combined with enhancement of birefringency, may be considered specific for collagen. An improved and modified method of staining with Sirius Red is presented.     

A simple micromethod for collagen and total protein determination in formalin-fixed paraffin-embedded sections

A simple, sensitive, and quantitative procedure is described for the measurement of collagen and protein content in tissue sections prepared from formalin-fixed paraffin-embedded samples. The method can detect as little as 5.7 micrograms of collagen per mg of protein. It is based on the selective binding of Sirius red F3BA and Fast green FCF to collagen and noncollagenous components, respectively, when the sections are stained with both dyes dissolved in aqueous saturated picric acid. Both dyes are eluted readily and simultaneously with NaOH-methanol and the absorbances obtained at 540 and 605 nm can be used to determine the amount of collagen and protein. The color equivalence of each dye was determined after destaining the sections and measuring the collagen content by hydroxyproline analysis and the amount of protein by the micro-Kjeldahl procedure. When several sections prepared from five rat tissues were analyzed first by the dye binding method and then by the chemical procedure, comparable results were obtained. This method could be of use in measuring collagen in tissue specimens and could be helpful in assessing the degree of fibrosis in tissue samples and in evaluating the effects of antifibrogenic drugs currently in use.     

Sensitive spectrophotometric method for the quantitative estimation of collagen

A method to estimate collagen concentration is described. It is based on the ability of the dye Sirius Red F3BA to quantitatively precipitate collagen in acetic acid solution. Collagen content is determined by evaluating spectrophotometrically the absorbance decrease of a Sirius Red/acetic acid solution. The assay can be used in the range from 1 to 50 micrograms protein. The method is specific, sensitive, simple, and rapid.     

Polarization microscopy and microspectrophotometry of Sirius Red, Picrosirius and Chlorantine Fast Red aggregates and of their complexes with collagen

Summary A detailed quantitative analysis of the anisotropic properties of Sirius Red F3B, Picrosirius, and Chlorantine Fast Red crystals, and of their complexes with a macromolecularly oriented protein either in a pure form or as part of a tissue structure was carried out. Collagen I was used as the protein model. Linear dichroism and dispersion of birefringence were investigated in dye aggregates, in stained filaments of collagen I and in collagen bundles in sections of tendon. A positive linear dichroism, the characteristics of which varied as a function of the dye type used, was demonstrated for the dye aggregates and stained substrates. However, even thin regions of the stained tendon collagen bundles showed very high absorbances, differing from the pattern reported previously, for collagen stained with another sulphonated azo dye, Xylidine Ponceau. Consequently, not all these dyes enable protein concentration and orientation to be determined in collagen-containing structures. From the linear dichroism patterns it is assumed that the long axis of the molecules of these azo dye is mostly parallel to that of filaments of pure collagen I and statistically parallel to the long axis of collagen bundles of tendon sections. The dye aggregates and, stained pure collagen I and tendon collagen bundles exhibited birefringent images with interference colours that varied as a function of thickness and packing state of the preparations, which is in agreement with reports in the literature. The optical retardations of the collagen bundles increased by a factor of 5–6 times after staining with Picrosirius. From data on form dichroism it is concluded that when studying the macromolecular orientation of collagen preparations stained with azo dyes, the choice of the mounting medium deserves consideration.     

Microplate reader-based quantitation of collagens.

By using a picrosirius dye, sensitive and specific staining of collagens plated in microtiter wells was achieved. The range of detection was from 0.5 to 20 micrograms. Human collagen types I, III, IV, and V were tested and able to be detected by the method. The dye did not bind to acetylcholinesterase or elastin. It did bind to C1q to some extent but this is not surprising since the molecule contains some triple helical collagen-like structures. A comparison performed between this assay and a colorimetric assay for hydroxyproline using tissue culture supernatants gave similar results for both samples. Due to its simplicity and sensitivity this assay will be most useful in laboratories where large numbers of samples must be screened for collagen production.     

One-bath trichrome staining: Investigation of a general mechanism based on a structure-staining correlation analysis

Summary Dye pairs of contrasting colours were selected from acid dyes of varied chemical characteristics. The 44 dye pairs were investigated in a one-bath trichrome staining system in which the dye-baths were strongly acid. Dye concentrations, concentration ratios and staining times were varied for each dye pair. Thirty dye pairs stained collagen fibres distinctly different colours to muscle cytoplasm, while 14 dye pairs gave muddy, non-selective staining. Comparison of dye structures showed that in selective pairs the larger dye always stained the collagen fibres, with cytoplasm being coloured by the smaller species. With 28/30 of the selective dye pairs the differences in anionic weights of the dyes was > 200. However, in dye pairs giving non-selective staining, the anionic weights of the members of 13/14 of the dye pairs differed by < 200. As no other structural feature correlated so clearly with selectivity, it was concluded that the selectivity of one-bath trichromes is diffusion-rate controlled, involving the interaction of differentially permeable tissue sites (collagen being more permeable than muscle cytoplasm) with dyes diffusing at different rates (large dyes slower than small). In keeping with this, lengthening staining times reduced staining selectivity. The rate control mechanism suggested a rational trouble-shooting guide for one-bath trichromes, encompassing such practical factors as dye concentration, embedding medium, fixative, dye-bath pH, section thickness and staining time.     

Supramolecular order following binding of the dichroic birefringent sulfonic dye Ponceau SS to collagen fibers

The optical anisotropies (linear dichroism or LD and birefringence) of crystalline aggregates of the sulfonic azo-dye Ponceau SS and of dye complexed with chicken tendon collagen fibers were investigated in order to assess their polarizing properties and similarity to liquid crystals. In some experiments, the staining was preceded by treatment with picric acid. Crystalline fibrous aggregates of the dye had a negative LD, and their electronic transitions were oriented perpendicular to the filamentary structures. The binding of Ponceau SS molecules to the collagen fibers altered the LD signal, with variations in the fiber orientation affecting the resulting dichroic ratios. The long axis of the rod-like dye molecule was assumed to be bound in register, parallel to the collagen fiber. Picric acid did not affect the oriented binding of the azo dye to collagen fibers. There were differences in the optical anisotropy of Ponceau SS-stained tendons from 21-day-old and 41-day-old chickens, indicating that Ponceau SS was able to distinguish between different ordered states of macromolecular aggregation in chicken tendon collagen fibers. In the presence of dichroic rod-like azo-dye molecules such as Ponceau SS, collagen also formed structures with a much higher degree of orientation. The presence of LD in the Ponceau SS-collagen complex even in unpolarized light indicated that this complex can act as a polarizer.     

Pleochroism in tendon and its bearing to acid mucopolysaccharides

Summary and conclusions The pleochroism found in the histological sections dyed with toluidine blue buffered at pH 4 is caused by the selective absorption of polarized light by the molecule of the dye. This fact makes it possible to calculate the position of the molecules of acid mucopolysaccharides (AMP) by indirect method.The molecules of AMP, of linear or bastonet shape are placed parallel to the long axis of the collagen fibers, with their negative polar groups directed toward the collagen.This structural relation between the molecules of the AMP and the collagen not only facilitate a cementing and stabilizing action but contribute to the resistance to traction as well.The author would like to thank Prof. Lucien Lison for his help and encouragement.     

碱性成纤维细胞生长因子与胶原对组织工程软骨体外构建的影响

目的：以三维成团培养为培养系统,探讨bFGF与胶原对组织工程软骨体外构建的影响。方法：成团培养兔生长板软骨细胞,设bFGF、胶原及联合作用组。HE染色观察新生组织形态;免疫组化检测Ⅰ、Ⅱ型胶原表达以观察细胞表型;Hoechst 33258法检测细胞DNA含量;羟脯氨酸法与阿新蓝法测定基质中胶原与蛋白多糖的合成。结果：新生软骨的组织学形态近似自然软骨;各实验组软骨细胞DNA含量明显上升;胶原可以显著促进基质的合成;各实验组Ⅰ型胶原的表达少于对照组,Ⅱ型胶原的表达则高于对照组;联合作用组效果更加明显。结论：三维的成团培养可以促进基质合成,有效维持软骨细胞表型;bFGF与胶原有利于工程化软骨构建,其效果具有协同效应,两者联合应用可进一步促进软骨再生。     

Use of a cyanine dye as a probe for albumin and collagen in the extracellular matrix

The aim of this work was to develop a quick method for analysis of macromolecules of the extracellular matrix. Of great interest are soluble components of the extracellular matrix, in particular, carrier proteins, whose variation dynamics can characterize the studied tissue in its development, adult stage, and aging. We suggest the method of analysis of the extracellular matrix to reveal the presence of albumin and collagen by using an anionic cyanine dye as a spectral and fluorescence probe. The method was applied for the analysis of the human vitreous body in the course of its development. Albumin was detected by the appearance of the trans monomer absorption and fluorescence bands in the dye spectra, and collagen was detected by the absorption and fluorescence bands of J aggregates. Hyaluronic acid present in the vitreous body does not interfere with the results of the analysis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis confirmed the presence of albumin in the vitreous body. We suppose that albumin as a protein carrying biologically active macromolecules plays an important role in the processes of differentiation and functional establishment of ocular tissues in the course of their prenatal development.     

Density quantification of collagen grafted on biodegradable polyester: its application to esophageal smooth muscle cell

An improved technique for quantification of collagen immobilized on polymeric substrates is needed as tissue engineering evolves. Current immobilized protein quantification methods are indirect, time-consuming, and/or inaccurate. In this study, Sirius red colorimetric microassay was shown to be feasible for quantifying the density of collagen immobilized on aminolyzed poly(L-lactic acid) (PLLA) surfaces using the specific bonding of Sirius dye to collagen. It offers a number of advantages over traditional methods, including direct staining, high sensitivity, and high stability of the dye. The detection limit is approximately 0.1 microg/cm(2), and the dynamic range is greater than 50. Sirius red dye has not been used previously for quantification of protein immobilized on polymers. The collagen densities achieved with each of the two crosslinking reagents investigated, namely glutaraldehyde (GA) and genipin, were compared. The latter is an alternative crosslinker derived from a traditional Chinese medicine. The collagen densities immobilized by the two reagents were measured to be similar. This was confirmed by the similar behaviors of esophageal primary smooth muscle cells (ESMCs) on these two modified PLLA membranes; collagen grafted with either coupler was found to greatly promote, to a similar extent, cell attachment and both short-term (4 days) and long-term (12 days) proliferation compared with unmodified PLLA. Smooth muscle cells on both modified membranes were stained to display contractile alpha-actin protein filaments.     

Simultaneous observation of collagen and elastin in normal and pathological tissues: analysis of Sirius-red-stained sections by fluorescence microscopy

In order to observe collagen and elastic fibers simultaneously, sections of human aorta, skin, lung, liver, and bladder were stained by Sirius red and analyzed by fluorescence microscopy. In all cases, the fibers of collagen presented the characteristic fluorescent red-orange color that results from the interaction of this extracellular protein with the dye, whereas elastic fibers showed strong green fluorescence (intrinsic fluorescence). This method efficiently detects collagen and elastic fibers when these two structures are present and could have valuable applications in processes that involves both fibers.L.F.B. received a doctoral fellowship from the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Brazil.     

Colorful Protein-Based Fluorescent Probes for Collagen Imaging

Real-time visualization of collagen is important in studies on tissue formation and remodeling in the research fields of developmental biology and tissue engineering. Our group has previously reported on a fluorescent probe for the specific imaging of collagen in live tissue in situ, consisting of the native collagen binding protein CNA35 labeled with fluorescent dye Oregon Green 488 (CNA35-OG488). The CNA35-OG488 probe has become widely used for collagen imaging. To allow for the use of CNA35-based probes in a broader range of applications, we here present a toolbox of six genetically-encoded collagen probes which are fusions of CNA35 to fluorescent proteins that span the visible spectrum: mTurquoise2, EGFP, mAmetrine, LSSmOrange, tdTomato and mCherry. While CNA35-OG488 requires a chemical conjugation step for labeling with the fluorescent dye, these protein-based probes can be easily produced in high yields by expression in E. coli and purified in one step using Ni²⁺-affinity chromatography. The probes all bind specifically to collagen, both in vitro and in porcine pericardial tissue. Some first applications of the probes are shown in multicolor imaging of engineered tissue and two-photon imaging of collagen in human skin. The fully-genetic encoding of the new probes makes them easily accessible to all scientists interested in collagen formation and remodeling.     

Synthetic Orcein as an Elastic Tissue Stain

A study was made of various synthetic orceins in an effort to determine the optimal conditions for their use in staining elastic tissue. A simple technic has been developed, based on a modification of the method originally worked out by Taenzer. Orcein is used as a 0.4% solution in 70% alcohol containing 1% HC1. Sections are counterstained with Mallory's borax methylene blue. The solutions are easily prepared and may be used for several months. Although several methods require staining in orcein from 2 to 24 hours, the present method requires staining for only 30 minutes. After several types of fixation some of the dye samples stained collagenous tissue to varying degrees, but this could be diminished by slightly reducing the strength of the staining solution. Differential staining of elastic tissue was particularly specific in tissues fixed in acetone, collagen in these preparations remaining practically unstained by any of the dye samples.     

High resolution imaging of collagen organisation and synthesis using a versatile collagen specific probe

Collagen is the protein primarily responsible for the load-bearing properties of tissues and collagen architecture is one of the main determinants of the mechanical properties of tissues. Visualisation of changes in collagen three-dimensional structure is essential in order to improve our understanding of collagen fibril formation and remodelling, e.g. in tissue engineering experiments. A recently developed collagen probe, based on a natural collagen binding protein (CNA35) conjugated to a fluorescent dye, showed to be much more specific to collagen than existing fluorescent techniques currently used for collagen visualisation in live tissues. In this paper, imaging with this fluorescent CNA35 probe was compared to imaging with second harmonic generation (SHG) and the imaging of two- and three-dimensional collagen organisation was further developed. A range of samples (cell culture, blood vessels and engineered tissues) was imaged to illustrate the potential of this collagen probe. This images of collagen organisation showed improved detail compared to images generated with SHG, which is currently the most effective method for viewing three-dimensional collagen organisation in tissues. In conclusion, the fluorescent CNA35 probe allows easy access to high resolution imaging of collagen, ranging from very young fibrils to more mature collagen fibres. Furthermore, this probe enabled real-time visualisation of collagen synthesis in cell culture, which provides new opportunities to study collagen synthesis and remodelling.     

Dermatan sulphate-rich proteoglycan associates with rat tail-tendon collagen at the d band in the gap region.

Rat tail tendon was stained with a cationic phthalocyanin dye, Cupromeronic Blue, in a 'critical-electrolyte-concentration' method [Scott (1980) Biochem. J. 187, 887-891] specifically to demonstrate proteoglycan by electron microscopy. Hyaluronidase digestion in the presence of proteinase inhibitors corroborated the results. Collagen was stained with uranyl acetate and/or phosphotungstic acid to demonstrate the banding pattern a-e in the D period. Proteoglycan was distributed about the collagen fibrils in an orthogonal array, the transverse elements of which were located almost exclusively at the d band, in the gap zone. The proteoglycan may inhibit (1) fibril radial growth by accretion of collagen molecules or fibril fusion, through interference with cross-linking, and (2) calcification by occupying the holes in the gap region later to be filled with hydroxyapatite.     

Ultrastructural localization of proteoglycans in tissue using Cuprolinic Blue according to the critical electrolyte concentration method: Comparison with biochemical data from the literature

Summary Several connective tissues were stained for proteoglycans using the cationic dye Cuprolinic Blue according to the critical electrolyte concentration method. With this method, proteoglycans are visualized as electron-dense filaments. In most tissues, two types of proteoglycan filaments are present: a small (maximum length 60 nm), thin, collagen fibril-associated filament, and a thick, heavily-staining filament which is predominantly localized between bundles of collagen fibrils. Cartilage contains very large (about 300 nm) proteoglycan filaments while in cornea they are very small. Comparison with biochemical data from the literature suggests that the appearance of the proteoglycan filaments may be indicative for the glycosaminoglycan—protein ratio and for the molecular weight of the part of the protein core to which glycosaminoglycans are attached. The data thus obtained on the localization and structure of a proteoglycan may be useful when planning a strategy for its isolation.     

Staining of macromolecules: possible mechanisms and examples

This review is based on a presentation given at the Biological Stain Commission meeting in June 2008. I discuss staining as an interaction between dye, solvent, and biological macromolecules. Most staining takes place in water, where the physico-chemical properties of the macromolecules are particularly important. Staining from aqueous solution is summarized. The first step is diffusion–ion exchange, which builds up the dye ion concentration close to the appropriately charged tissue constituents. While charge interactions are important for selectivity and build-up of dye ions around specific tissue and cell constituents, they have in most cases little to do with actual dye binding. The next step, actual binding, is predominantly between aromatic and other non-polar parts of the dye and corresponding groups in the tissue constituent. This results in a reduction of the total hydrophobic area exposed to water, hence the term hydrophobic interaction. Because dye binding is predominantly by dispersive forces, the larger the aromatic dye system and the fewer the number of charges on the dye, the greater the substantivity or affinity. Some relatively straightforward anionic or cationic one-step staining systems are discussed also. These include amyloid staining with Congo red, elastin staining with orceins, collagen staining with picrofuchsin, DNA–RNA staining with methyl green-pyronin Y, acid heteroglycan staining with Alcian blue, and metachromatic staining.     

Measurement of collagen-proteoglycan interaction in hypertrophic scars

Collagen-proteoglycan interaction in hypertrophic scars was measured by staining tissue collagen with Sirius red, followed by elution of the bound stain, and photometric quantitation before and after papain digestion. Control sections from each of the specimens were treated with buffer solution instead of papain. The difference in dye concentration observed between respective untreated and digested sections was considered to be due to the unmasked basic groups of collagen originally bound to proteoglycans. The results suggest a significantly increased collagen-proteoglycan interaction in human hypertrophic scars as compared with nonhypertrophic scars, normal skin, and granulation tissues.     

Are picro-dye reactions for collagens quantitative? Chemical and histochemical considerations

Previous studies of picro-dye reactions demonstrated wide variations in the binding of different dyes. Picro-Sirius Red F3BA was recommended because it colors all collagens intensely and is suitable for polarization microscopy. Recent publications on quantitative uses of this stain were surprising. To obtain further information on the chemical mechanisms of dye binding by proteins, 94 sulfonated azo dyes were tested under the conditions of the picro-Sirius Red F3BA reaction. Reaction patterns varied widely, from failure to compete successfully with picrate ions for binding sites to strong coloration of all tissue structures. Only a few dyes stained collagen, reticulum fibers and basement membranes intensely and selectively. The reactivity of dyes was determined by their molecular configuration and the nature and position of substituents. Correlation with physico-chemical data showed that dye binding is due to non-ionic interactions, i.e. van der Waals and dispersion forces and hydrophobic bonding. Coulomb forces do not impart affinity - increasing sulfonation actually decreases dye uptake - but draw dyes within reach of non-ionic sites. Bound dyes form aggregates with additional dye ions; the aggregation number can range from 2 to many powers of 10. Clearly, dye binding by proteins is not stoichiometric.