The demonstration of bone and cartilage remodelling using alcian blue and hematoxylin

A new staining technique based on alcian blue and hematoxylin was used during the study of experimentally produced fractures in long bone. The distinction between cartilage, woven bone, mature bone and necrotic bone during successive stages of the fracture healing process was particularly remarkable. This method was found also to be very useful in the study of general tissue morphology. It is suggested that this postdecalcification light microscopy staining method is suitable for studies of cartilage and bone development with particular reference to ossification, remodelling and bone pathology.     

Energy dispersive X-ray microanalysis of sulfated glycosaminoglycans in cartilage matrix stained with alcian blue 8GX

X-ray spectra were recorded from 400-700 nm matrix areas of 0.5 micron sections prepared from the articular cartilages of 15- and 23-year-old human cadavers. The X-ray microanalysis was carried out (i) on untreated material; (ii) after removing sulfate group by a methylation procedure; (iii) after staining with a copper containing cationic phatolcyanin dye, alcian blue 8GX, preceded by carboxymethylation. K alpha peaks of sulphur could be detected in methylated (i.e. desulfated) samples. These peaks probably indicated the presence of sulphur-containing amino acids in different matrix proteins. Consequently, the measurements of sulphur despite its general use cannot be recommended for the X-ray microanalysis of sulfated glycosaminoglycans of cartilage matrix. K alpha peaks of copper could be identified after carboxymethylation and staining with alcian blue. After carboxymethylation, alcian blue can only be bound to the dissociated sulfate groups of glycosaminoglycans in the cartilage matrix. According to our spectrophotometric studies, approximately one molecule of alcian blue combined with one sulfate group. These data suggested that this technique could be used for semiquantitative estimation of sulfated glycosaminoglycans in small areas of the cartilage matrix. Using this method, we found a higher occurrence of sulfated glycosaminoglycans in the territorial matrix than in the interterritorial matrix of the intermediate and deep zones of the human articular cartilage.     

Differential staining of cartilage and bone in whole mouse fetuses by alcian blue and alizarin red S

The procedure described by Inouye ('76) for the staining of full-term mouse fetal skeletons has been adapted for use with mouse embryos and fetuses of days 14-18 of gestation. The main adaptations for younger specimens involve a longer time in acetone, in lieu of skinning, and omission of the aqueous KOH step. These adaptations require more time but result in consistently good staining of intact specimens.     

Improved demonstration of mast cells using alcian blue tetrakis (methylpyridium) chloride

Many batches of alcian blue dye are incompletely soluble at the low pH used for demonstrating mast cells. An improved technique using alcian blue tetrakis (methylpyridium) chloride (alcian blue pyridine variant) is described here. It produces stronger mast cell staining than other alcian blue stains tested.     

A procedure for differential staining of cartilage and bone in whole formalin-fixed vertebrates.

This paper describes a modification of the Simons and Van Horn (1971) procedure for rendering cartilage blue, bone red, and soft tissue translucent or transparent in whole vertebrate specimens. Alcian blue and alizarin red S are used to stain cartilage and bone respectively. In our procedure formalin is used as a fixative. This is a significant modification because formalin is the common fixative for museum specimens. This clearing and staining procedure is thus readily applicable to comparative studies in anatomy, embryology and systematic zoology.     

Improved demonstration of mast cells using alcian blue tetrakis (methylpyridium) chloride

Many batches of alcian blue dye are incompletely soluble at the low pH used for demonstrating mast cells. An improved technique using alcian blue tetrakis (methylpyridium) chloride (alcian blue pyridine variant) is described here. It produces stronger mast cell staining than other alcian blue stains tested.     

A modified differential stain for cartilage and bone in whole mount preparations of mammalian fetuses and small vertebrates

Fixation in formol-acetic-alcohol as a prelude to the staining of whole mount vertebrate skeletons with alcian blue and alizarin red S has greatly facilitated the enzyme clearing step of the method outlined by Dingerkus and Uhler. The modified method has been tested on fetal and neonatal mice, and on a variety of vertebrates including bony fish, reptiles, amphibia and birds, and shown to be rapid, reproducible and permanent. The method is not so rapid as that reported by Kimmel and Trammell but is superior at least in certain circumstances. In the present study, optimal results were obtained by fixing in formol-acetic-alcohol for 40 minutes, staining cartilage with alcian blue 8GX, then clearing with trypsin. The time taken to complete the latter step was reduced significantly by incubation at 37 C. The next step was to stain bone using alizarin red S in a weak solution of potassium hydroxide, followed by clearing in a potassium hydroxide-glycerol series.     

Freeze-substitution staining of rat growth plate cartilage with alcian blue for electron microscopic study of proteoglycans

To selectively stain polyanionic macromolecules of growth plate cartilage and to prevent artifacts induced by aqueous fixation, proximal tibial growth plates were excised from rats, slam-frozen, and freeze-substituted in 100% methanol containing the cationic dye Alcian blue. Electron microscopic examination showed the tissue stained with Alcian blue to be comparable in ultrastructural preservation to tissues slam-frozen and freeze-substituted in the absence of Alcian blue. The extracellular matrix exhibited a characteristic staining pattern when stained by this method. The pericellular rim was identified as a band of varying width encircling the chondrocyte and its cell processes. Peripheral to the pericellular rim the heterogeneity of staining within the extracellular matrix increased, taking the form of polymorphic densities. X-ray microanalysis showed that the visual interpretation of electron density was related to the concentration of copper present, and that the concentration of sulfur was variable in the pericellular rim and in the interterritorial matrix. The difficulties associated with aqueous fixation and staining procedures are discussed in contrast to the improved preservation achieved by cryogenic methods.     

Automated differential staining for cartilage and bone in whole mount preparations of vertebrates

An automated, rapid procedure for differential staining of cartilage and bone of vertebrates is described. The process involves rapid, complete staining of freshly skinned, eviscerated specimens after 30 sec immersion in a 70 C water bath, fixation in formol acetic alcohol and a rinse in 70% alcohol. Using an automatic tissue processor, the specimen is stained in alcian blue for 24 hr and macerated in 3% potassium hydroxide for 8 hr. Staining in alizarin red with maceration in 3% potassium hydroxide is completed manually. The specimens are cleared and preserved in glycerol. Good quality evenly stained specimens can be examined in less than three days and up to 600 fetuses can be processed in less than five days.     

The use of alcian blue in vivo

Summary Parenteral use of Alcian Blue in rabbits leads to apparent staining of glomerular basement membrane acutely but there is a particularization of the dye in mesangial structures and endothelial cells during the second to fourteenth day following injection. Membranous structures in pancreas, stomach and small intestine are also colored, possibly stained. The effect in the kidney and perhaps in other structures may be a non-specific one, based on filtration of the dye through such a naturally positioned barrier as basement membrane.A comparison of in vivo and in vitro staining patterns showed marked similarity in the deposition of the dye, suggesting specificity of the in vivo staining pattern. But the alteration of staining patterns with time could be construed as arguing against specificity.In the generalized Shwartzman reaction, Alcian Blue stained glomerular occlusive deposits, lending credence to, but not proving, the hypothesis that an acid mucopolysaccharide, with altered fibrinogen, may be implicated in the formation of glomerular aggregates in the Shwartzmann phenomenon.Supported by Research Grant AM 05071, United States Public Health Service, Bethesda, Md., and by the Health Research Council of the City of New York, of which the authors are Career Scientists.     

An easy technique for the clearing of histochemically stained plant tissue

The sites of GUS-activity in strongly pigmented whole-mount preparations or thick handmade sections of plant material can easily be visualized by clearing it with chlorallactophenol (CLP), without affecting the ClBr-indigo precipitate.     

On the stainability of plant cell walls with alcian blue

This work is a continuation of a communication on the stainability of broad bean (Vicia faba L.) root tip cells with alcian blue, published some time ago. Following the standard method of staining with alcian blue, the cell walls are very strongly stained, the nuclei (except nucleolus) lightly, the nucleolus and cytoplasm are practically colourless. The weak dyeing of the nucleus is not equal throughout the whole section so that the comparison of stainability of cell walls and nuclei by itself cannot explain the staining with alcian blue. The results of this work on the staining of cell walls (if not including model experiments and experiments in vitro, which are not considered as decisive here) can be summarized as follows: the pH dependence of staining, the loss of stainability as a result of pectinase digestion, blocking of staining by methylation and regeneration of stainability by demethylation and, finally, the impossibility of staining in the presence of NaCl lead to the conclusion that the staining of the material studied in this work is primarily caused by the salt linkage of alcian blue with the free carboxyls of pectic substances. From the comparison of staining with alcian blue and with other basic dyes it follows that in the case of alcian blue some other factors may also take part and are the reason for the selectivity and firmness (fastness) of the staining of cell walls with this dye. Otherwise, the staining of plant cell walls with alcian blue corresponds quite well to the staining of carboxyls containing polysaccharides of animal tissues with this dye. By staining with alcian blue it was found impossible to distinguish between younger and older cell walls within the meristem. However, this staining is suitable for routine use when studying the meristematic tissue. It is often possible to use solutions of a higher pH than generally used.     

On the blue coloration of vertebrates

Although the various vertebrate classes, from fishes to mammals are each distinctive, they possess many common features making it important to understand their comparative biology. One general feature that has long commanded interest is the integumental pigmentary system. Thus, much is known about particular pigment cells; however, the basis for some specific colors, such as blue, has escaped the scrutiny of the comparative approach. Regardless of Class, blue is almost always a structural color based upon incoherent or coherent scatter of blue wavelengths from the animal surface. The source of scatter may be intracellular or extra-cellular. A main intracellular scatterer is the surface of reflecting platelets of iridophores of lower vertebrates. Extra-cellular scatter is widespread and thought to occur from ordered dermal collagen arrays in primitive fishes, birds and mammals including humans. Among birds, feather structures provide major means for extra-cellular light scatter. There is only one known example of blue color deriving from a blue pigment found within a pigment cell. For amphibians, reptiles and birds, the scatter of blue wavelengths, together with the presence of yellow pigmentation, is fundamental for the expression of green coloration.     

Atlas of rat fetal skeleton double stained for bone and cartilage

BACKGROUND: The double staining of fetal skeleton for bone and cartilage is a very useful method to evidence skeletal abnormalities in laboratory animals. However, this method has been rarely used in routine developmental toxicity tests. One reason could be the difficulty of comparing the single skeletal pieces and of having reference points. In this paper the fetal rat skeleton double stained with Alizarin red S and Alcyan Blue is described in detail to produce an atlas for developmental toxicity laboratories.