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.     

Histochemical Detection of Collagen Fibers by Sirius Red/Fast Green Is More Sensitive than van Gieson or Sirius Red Alone in Normal and Inflamed Rat Colon

Collagen detection in histological sections and its quantitative estimation by computer-aided image analysis represent important procedures to assess tissue localization and distribution of connective fibers. Different histochemical approaches have been proposed to detect and quantify collagen deposition in paraffin slices with different degrees of satisfaction. The present study was performed to compare the qualitative and quantitative efficiency of three histochemical methods available for collagen staining in paraffin sections of colon. van Gieson, Sirius Red and Sirius Red/Fast Green stainings were carried out for collagen detection and quantitative estimation by morphometric image analysis in colonic specimens from normal rats or animals with 2,4-dinitrobenzenesulfonic acid (DNBS) induced colitis. Haematoxylin/eosin staining was carried out to assess tissue morphology and histopathological lesions. Among the three investigated methods, Sirius Red/Fast Green staining allowed to best highlight well-defined red-stained collagen fibers and to obtain the highest quantitative results by morphometric image analysis in both normal and inflamed colon. Collagen fibers, which stood out against the green-stained non-collagen components, could be clearly appreciated, even in their thinner networks, within all layers of normal or inflamed colonic wall. The present study provides evidence that, as compared with Sirius Red alone or van Gieson staining, the Sirius Red/Fast Green method is the most sensitive, in terms of both qualitative and quantitative evaluation of collagen fibers, in paraffin sections of both normal and inflamed colon.     

Histophotometric estimation of volume density of collagen as an indication of fibrosis in rat liver

The development of fibrosis in the liver of 16 rats treated for 1, 2, 3 or 4 weeks with CCl4, has been followed with chemical hydroxyproline determination and histophotometric analysis of histological sections stained with Sirius Red F3BA in saturated aqueous picric acid. The readings were taken with a scanning and integrating microphotometer and corrected for picric acid absorbance as a measure for mean protein mass per unit area of the section. It appears that the integrated absorbance readings of Sirius Red absorbing material in the section show a highly significant correlation with the hydroxyproline determinations. It is concluded that picrosirius photometry can be used to give a measure of the volume density of collagen in sections. An advantage of the photometric assay is that measurements are taken on the basis of the microscopic image, so that it is also possible to estimate collagen density in a selected area, e.g. a tumour formation amidst normal tissue, or to exclude necrotic areas.     

Hostophotometric estimation of volume density of collagen as an indication of fibrosis in rat liver

Summary The development of fibrosis in the liver of 16 rats treated for 1, 2, 3 or 4 weeks with CCl₄, has been followed with chemical hydroxyproline determination and histophotometric analysis of histological sections stained with Sirius Red F3BA in saturated aqueous picric acid. The readings were taken with a scanning and integrating microphotometer and corrected for picric acid absorbance as a measure for mean protein mass per unit area of the section. It appearts that the integrated absorbance readings of Sirius Red absorbing material in the section show a highly significant correlation with the hydroxyproline determinations. It is concluded that picrosirius photometry can be used to give a measure of the volume density of collagen in sections. An advantage of the photometric assay is that measurements are taken on the basis of the microscopic image, so that it is also possible to estimate collagen density in a selected area, e.g. a tumour formation amidst normal tissue, or to exclude necrotic areas.     

Preparation of ready-to-use, storable and reconstituted type I collagen from rat tail tendon for tissue engineering applications

Collagen is a widely investigated extracellular matrix material with extensive potentials in the field of tissue engineering. This protocol describes a method to prepare reconstituted collagen that can be ready-to-use, storable and suitable for further in vitro and in vivo investigations. Type I collagen was extracted from rat tail tendons and processed in acetic acid solution to obtain sterile soluble collagen. At first, crude collagen was dissolved in acetic acid, then frozen at -20 degrees C and lyophilized to obtain a sponge, which could be stored at -80 degrees C. Lyophilized collagen was then dispersed in acetic acid to obtain a sterile solution of collagen at targeted concentrations. The whole low-cost process from the extraction to the final sterile solution takes around 2-3 weeks. The collagen solution, once neutralized, has the potential to be used to produce gels or scaffolds, to deposit thin films on supports and to develop drug delivery systems.     

In situ measurement of collagen synthesis by human bone cells with a Sirius Red-based colorimetric microassay: effects of transforming growth factor β2 and ascorbic acid 2-phosphate

Staining of collagens by Sirius Red, a standard histological procedure, was applied to quantify collagen synthesis in human osteoblast-like cell cultures in situ. After morphological analysis of the deposited material, the stain was dissolved and its optical density determined spectrophotometrically using a microtiter plate assay system. The method was sensitive with a detection limit for collagen synthesized by 3000 normal human periosteal cells. The assay is easy to perform and specific with respect to different extracellular materials, for example, collagen types I and III were well stained, collagen type IV and laminin exhibited only low staining, and fibronectin, chondroitin sulfate, dermatan sulfate, and amyloid β were negative. A major advantage of the method is the combination of identification of collagen-producing cells in situ with subsequent spectrophotometric quantification of the dissolved stain. Thus it is possible to obtain information on cell morphology, active sites of collagen deposition in a cell culture, microscopic detection of high-and low-producer cells prior to dissolution and quantification of the deposited material. In this regard the assay is superior to either radioactive labeling, hydroxyproline determination, or Sirius Red-based colorimetric assays with cell lysates. Since the quantification is based on microtiter plate reading, the method can be recommended for the screening of large quantities of samples. Accepted: 30 June 1999     

A simple and sensitive method for the quantitative estimation of collagen.

A method to quantitate collagen in solution is described. It is based on binding of the dye Sirius Supra red F3BA by collagen followed by elution and estimation of the bound dye in a spectrophotometer. The assay can be used in the range from 10 to 100 μg protein. The method is rapid, specific, simple, sensitive, and highly reproducible.     

Microcalorimetric and electron spin resonance study of the influence of UV radiation on collagen

It has been shown by microcalorimetry that UV-irradiation cardinally alters the temperature dependence of heat capacity of a collagen solution and decreases the enthalpy of collagen heat denaturation. By using the method of electron spin resonance (ESR), it was found that the primary products of UV-irradiated acid-soluble collagen are the atomic hydrogen and the anion radical of acetic acid. The latter, under the influence of long-wavelength UV light, is transformed into the methyl radical, which interacts with acetic acid to produce acetic acid radical. The above free radicals interact with the collagen molecule, as a result of which seven superfine components with the split of deltaH = 1.13 mT are obtained in the ESR spectrum. It is assumed that this spectrum is related to the free radical that occurred in the proline residue of the collagen molecule. In this particular case, this is a major structural defect in the triple helix of collagen, which results in instability of the macromolecule.     

Cyclooxygenase-2 expression in skeletal muscle of knockout mice suffering Duchenne muscular dystrophy

The purpose of the present study was to investigate the role of cyclooxygenase-2 (COX-2) expression in fibrotic lesion in mdx mice. A total of six male C57BL/10 mice and six C57BL/10-DMD/mdx were distributed into two groups: control and animals with Duchenne muscular dystrophy (DMD). The medial part of gastrocnemius muscle was evaluated being the specimens stained with hematoxylin and eosin (H&E) and Sirius Red under normal and polarized light to differentiate type I (red and yellow) and III (green) collagen. COX-2 expression was assessed by immunohistochemistry. The results revealed histopathological changes in C57BL/10-DMD/mdx as depicted by regenerating fibers. Sirius Red stain showed a substantial increase in the amount of type I collagen of mdx mice. DMD induced a strong COX-2 immunoexpression in intercellular space. Taken together, our results are consistent with the notion that necrotic and fibrotic lesions are able to increase COX-2 expression in DMD.     

Preservation of elastic system fibers and of collagen molecular arrangement and stainability in an Egyptian mummy

The present findings show that both elastic system fibers and collagen markedly resisted change in tissues more than 2000 years old. The distribution of elastic fibers and elastic-related fibers (namely, oxytalan and elaunin fibers) in mummified tissues coincided with the observations made on the modern human tissues used as controls. The collagenous structures present in tissue sections obtained from the Egyptian mummy studied took on a deeply red colour when stained in the Picrosirius solution indicating that, as well as in the fresh controls, the basic groups in the collagen molecules were available for reacting with the strongly acidic dye Sirius Red. When viewed with polarized light, the collagen in the same tissue sections displayed an increased birefringence, which shows that the collagen molecules in mummified tissues maintain the oriented disposition which is typical of the modern human tissues used as controls. The methods employed have proved to be useful for the delineation of the elastic system fibers and of the collagenous scaffolding, which may be used as valuable landmarks in the study of the histoarchitecture of organs that have undergone considerable distortion.     

Method of addition of acetonitrile influences the structure and stability of collagen

Collagen has been extensively used as a biomaterial in many biomedical applications. Recently, collagen based biomaterials were prepared using organic solvents. In this context, the method of addition of organic solvent described in the present study will be an important contribution in the preparation of collagen-based biomaterials. The effect of acetonitrile on collagen structure and stability was investigated using biophysical methods. Collagen undergoes solvent-induced denaturation with increasing concentration of acetonitrile. It was observed that addition of acetonitrile (50–90%) to a collagen solution in a single shot (method 1) led to precipitation. Contrary, collagen remained in the solution when acetonitrile content was increased to 90% in a collagen solution that had been formerly equilibrated with 20% acetonitrile (method 2). Interestingly, triple helical structure was retained when precipitated collagen, obtained from method 1, was re-dissolved in acetic acid solution. The re-dissolved collagen exhibits comparable melting temperature as that of native collagen. Re-dissolved collagen also showed fibril formation, but with decreased rate. The soluble collagen in 90% acetonitrile, prepared by method 2, is found to be unordered. The above results thus suggest that the method of addition of acetonitrile plays an important role in the folding and unfolding of collagen.     

Modified Whipf's Polychrome: A Connective Tissue Stain with Special Application for Demonstrating Leishmania

A polychrome stain procedure was developed to demonstrate amastigotes of the protozoan parasite Leishmania braziliensis as well as cytoplasmic and other tissue components in cutaneous lesions of infected animals. The procedure is as follows: stain nuclei for 10 minutes with an iron hematoxylin containing 0.5% hematoxylin and 0.75% ferric ammonium sulfate dissolved in 1:1 0.6 N H₂SO₄:95% ethanol; rinse 4 minutes in distilled water. Cytoplasmic staining is achieved by exposing tissues for 10 minutes to a solution containing 0.25% Biebrich scarlet, 0.45% orange G, 0.5% phosphomolybdic acid and 0.5% phosphotungstic acid in 1% aqueous acetic acid. These first two solutions are modified from Whipf's polychrome stain. Sections are differentiated for 10 seconds in 50% ethanol, rinsed in water, stained 3 minutes in 0.1% aniline blue WS in saturated aqueous picric acid, rinsed in water and differentiated for 1 minute in absolute ethanol containing 0.05% acetic acid. Mordanting overnight in 6% picric acid in 95% ethanol produced optimal results.

This procedure was applied to sectioned material from experimental animals with various protozoa. Trypanosoma cruzi, Besnoitia Jellisoni, Toxoplasma gondii and especially Leishmania braziliensis were well demonstrated. Combining cytoplasmic dyes and phosphomolybdic-phosphotungstic acids into one solution afforded differential staining of tissues by Biebrich scarlet and orange G; connective tissues were stained by this solution. Substantially improved definition of connective tissues resulted after counterstaining. This procedure differs from the Massou sequence in which connective tissues are first stained by cytoplasmic dyes along with other tissues and then destained prior to specific counter-staining. in comparing dyes structurally related to Biebrich scarlet, it was found that Crocein scarlet MOO, but not Poncenu S, was an acceptable substitute. Sirius supra blue GL and Sirius red FSBA were not useful as replacements for aniline blue WS in this procedure.     

Preservation of elastic system fibers and of collagen molecular arrangement and stainability in an Egyptian mummy

Summary The present findings show that both elastic system fibers and collagen markedly resisted change in tissues more than 2000 years old.The distribution of elastic fibers and elastic-related fibers (namely, oxytalan and elaunin fibers) in mummified tissues coincided with the observations made on the modern human tissues used as controls.The collagenous structures present in tissue sections obtained from the Egyptian mummy studied took on a deeply red colour when stained in the Picrosirius solution indicating that, as well as in the fresh controls, the basic groups in the collagen molecules were available for reacting with the strongly acidic dye Sirius Red. When viewed with polarized light, the collagen in the same tissue sections displayed an increased birefringence, which shows that the collagen molecules in mummified tissues maintain the oriented disposition which is typical of the modern human tissues used as controls.The methods employed have proved to be useful for the delineation of the elastic system fibers and of the collagenous scaffolding, which may be used as valuable landmarks in the study of the histoarchitecture of organs that have undergone considerable distortion.Supported in part by Grant no. 43.83.0610/00 from Financiadora de Estudos e Projetos (FINEP-FNDCT). G.S. Montes is Career Investigator of the Brazilian National Research Council (CNPq)     

A microcalorimetric and electron spin resonance study of the influence of UV radiation on collagen

It was demonstrated microcalorimetrically that UV radiation cardinally changed the behavioral pattern of the temperature dependence of heat capacity of a collagen solution and decreased the enthalpy of collagen denaturation. It was discovered by electron spin resonance (ESR) that the primary products of UV-irradiated acid-soluble collagen were atomic hydrogen and the anion radical of acetic acid. The latter, when exposed to long-wavelength UV light, converted into the methyl radical, which interacted with acetic acid to produce the acetic acid radical. These free radicals interacted with the collagen molecule, leading to the appearance of seven superfine components with the split ΔH = 1.13 mT in the ESR spectrum. It was assumed that this spectrum is related to the free radical that occurred in the proline residue of the collagen molecule, which, in this particular case, was the major defect in the triple helix of collagen that caused instability of the macromolecule.     

Combining Immunodetection with Histochemical Techniques: The Effect of Heat-induced Antigen Retrieval on Picro-Sirius Red Staining

Picro-Sirius red is a routine diagnostic stain intended for the histological visualization of collagen fibers (fibrosis) in tissue. Multi-label immunohistochemistry is a powerful tool used by researchers to visualize different cell types and their location within a tissue specimen, and to observe co-localization of antigens. Combining the specificity of immunodetection with the simplicity of Sirius red staining will allow researchers to visualize multi-antigen detection in relation to fibrosis, a common histological feature of injury in many chronic diseases. Pre-treatment of formalin-fixed, paraffin-embedded tissue (FFPE) specimens with antigen retrieval is essential for the work-up of most commercially available antibodies. The most common form of antigen retrieval involves boiling tissue specimens in buffer to break the cross-linkages caused by formalin fixation. However, this method causes tissue modification and collagen fiber shrinkage leading to suboptimal results when counterstaining for Sirius red. Reduced heat and enzymatic digestion are antigen retrieval methods compatible with Sirius red counterstaining. This paper will discuss the difficulties faced when combining these two staining methods, and provide a detailed method for the simultaneous detection of antigen and Sirius red in FFPE tissues.     

鼠尾胶原蛋白提取、分离、纯化方法的建立及鉴定

目的建立一种高效提取、分离、纯化鼠尾胶原蛋白的方法。方法通过对鼠尾进行剥离获得鼠尾腱,用Tris-HCl缓冲液、胃蛋白酶处理获得鼠尾胶原蛋白原液、反复使用氯化钠溶液进行分级盐析、醋酸溶液复溶进行鼠尾胶原蛋白的纯化。超纯水透析除去无机盐类获得纯化的鼠尾胶原蛋白。通过SDS-PAGE蛋白质电泳、氨基酸含量分析等技术手段鉴定。结果本研究建立的方法可以获得高纯度的鼠尾胶原蛋白,纯度达到电泳纯。与国外进口的商业化鼠尾胶原蛋白产品相比无差异。研究了提取、分离、纯化参数对得率、纯度的影响,建立了最优的鼠尾胶原蛋白提取条件,胃蛋白酶用量：1∶500,酶解时间：72 h,盐析浓度：2 mol/L,提取所用酸溶液：0.05mol/L醋酸溶液。结论为鼠尾胶原蛋白的扩大化生产提供了合适的工艺参数,为大量获得鼠尾胶原蛋白并进行更深层次的功效方面研究提供了理论支持和实践基础。     

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.     

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.     

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.     

Effect of exogenous embryonic fibroblasts on ratio of collagens types I and III in tissues of mouse paraprosthetic capsule

At present, the use of synthetic prostheses is an obligatory condition of surgical treatment of extensive and giant ventral hernias. We have carried out a comparative investigation of the dynamics of the collagen Type I to Type III ratio in the mouse paraprosthetic region upon use of the implants Esphyl (polyethylene), Ecophlon (polytetrafluorethylene), and Uniflex (polyvinylidenfluoride) with or without single and twofold introduction of cultured fibroblasts into paraprosthetic region at different time periods. Use of staining with Sirius Red and polarization microscopy revealed that, at a short time period (10 days after endoprosthesis implantation), the fibroblast introduction and the multiplicity of their introduction do not affect the collagen type ratio. At later time periods (30–60 days after the endoprosthesis implantation), the collagen type I ratio increased with use of all materials. At all steps of the experiment, the collagen type I/III ratio was the highest in the case of use of the Uniflex endoprosthesis. The exogenous fibroblasts accelerate the increase in the collagen type I/III ratio to a greater degree during twofold than upon single introduction.