Detection of Sperm Histone Diversity Among Vertebrates by Alkaline Fast Green Staining

Testis sections from fifteen species from six classes of vertebrates were stained with alkaline fast green (AFG) to correlate staining differences with the known biochemical diversity of histones in the spermatozoa. After trichloroacetic acid (TCA) hydrolysis the sperm of some species known to contain sperm-specific histones did not stain. This correlation held if fixation in neutral buffered formalin was limited to 3 to 6 hr and hydrolysis was done at 90 C. The species whose sperm did stain after TCA hydrolysis could be divided into three groups. In some species the sperm no longer stained if, after TCA, the sections were treated with thioglycollic acid. These sperm contained basic proteins that were rich in cysteine. In turn, the group of species whose sperm continued to stain after TCA and thioglycollic acid treatments could be subdivided. The sperm of some were stained specifically without DNA hydrolysis if the AFG was made up with sodium chloride. These sperm contained sperm-specific histones. In other species the sperm did not stain under these conditions, and these sperm had a basic protein complement similar to that found in somatic cell nuclei. These correlations suggest that AFG staining can be used to detect sperm histone diversity in a wide range of organisms.     

HISTONES AND ALKALINE FAST GREEN STAINING OF ONION ROOTS

Onion root tips were freeze killed or fixed in 12 different chemical fixatives to determine the possible effects of such treatments on subsequent staining with alkaline Fast green (pH 8.0-8.1) to reveal the presence of histones. Ethanol, methanol, neutral formalin, and a mixture of methanolchloroform-acetic acid produced nearly comparable results. Subsequent to fixation or freeze drying the sections were treated with hot 5% trichloroacetic acid (TCA) to remove nucleic acids prior to Fast green staining. Staining of histones does not occur in chemically fixed material if TCA hydrolysis is omitted. Nuclei, chromosomes during mitosis, and cytoplasm (to varying degrees) were stainable after TCA treatment. Positive staining occurs in nuclei, nucleoli, and cytoplasm in freeze-dried material with or without prior TCA treatment. Comparisons are made with chemical fixations. The use of ribonuclease instead of TCA permits staining of the cytoplasm and of chromosomes during mitosis. Discussions are included on the stainability of nucleoli and the possibility that native histone is associated with ribonucleic acid (RNA).     

The chemical composition of nuclei and chromosomes isolated by the Langmuir trough technique

The pattern of staining for DNA, histone, and nonhistone protein has been studied in whole cells and in nuclei and chromosomes isolated by surface spreading. In whole interphase cells from bovine kidney tissue culture, nuclear staining for DNA and histones reveals numerous small, intensely stained clumps, surrounded by more diffusely stained material. Nuclei in whole cells stained for nonhistone proteins also contain intensely stained regions surrounded by diffuse stain. These intensely stained regions also stain for RNA, indicating that the regions contain nucleolar material. Electron microscopy of kidney cells confirms that multiple nucleoli are present. Kidney nuclei isolated by surface spreading show an even distribution of stain for DNA, histones, and nonhistone proteins, indicating that the surface forces disperse both condensed chromatin and nucleoli. DNA and protein staining was also studied in metaphase chromosomes from testes of the milkweed bug, Oncopeltus fasciatus. Staining for DNA and histones in metaphase chromosomes is essentially the same in sections of fixed and embedded testes as in preparations isolated by surface spreading. However, striking differences are noted in the distribution of nonhistone proteins. In sections, nonhistone stain is concentrated in extrachromosomal areas; metaphase chromosomes do not stain for nonhistone proteins. Chromosomes isolated by surface spreading, however, stain intensely for nonhistone proteins. This suggests that nonhistone proteins are bound to the chromosomes as a contaminant during the isolation procedure. The relationship of these findings to current work with chromosomes isolated for electron microscopy is discussed.     

The chemical composition of nuclei and chromosomes isolated by the Langmuir trough technique

The pattern of staining for DNA, histone, and nonhistone protein has been studied in whole cells and in nuclei and chromosomes isolated by surface spreading. In whole interphase cells from bovine kidney tissue culture, nuclear staining for DNA and histones reveals numerous small, intensely stained clumps, surrounded by more diffusely stained material. Nuclei in whole cells stained for nonhistone proteins also contain intensely stained regions surrounded by diffuse stain. These intensely stained regions also stain for RNA, indicating that the regions contain nucleolar material. Electron microscopy of kidney cells confirms that multiple nucleoli are present. Kidney nuclei isolated by surface spreading show an even distribution of stain for DNA, histones, and nonhistone proteins, indicating that the surface forces disperse both condensed chromatin and nucleoli. DNA and protein staining was also studied in metaphase chromosomes from testes of the milkweed bug, Oncopeltus fasciatus. Staining for DNA and histones in metaphase chromosomes is essentially the same in sections of fixed and embedded testes as in preparations isolated by surface spreading. However, striking differences are noted in the distribution of nonhistone proteins. In sections, nonhistone stain is concentrated in extrachromosomal areas; metaphase chromosomes do not stain for nonhistone proteins. Chromosomes isolated by surface spreading, however, stain intensely for nonhistone proteins. This suggests that nonhistone proteins are bound to the chromosomes as a contaminant during the isolation procedure. The relationship of these findings to current work with chromosomes isolated for electron microscopy is discussed.     

CHANGES IN NUCLEAR HISTONES DURING FERTILIZATION, AND EARLY EMBRYONIC DEVELOPMENT IN THE PULMONATE SNAIL, Helix aspersa

Calf thymus histories comprising two fractions, one rich in lysine, the other having roughly equal amounts of lysine and arginine, Loligo testes histones rich in arginine, and salmine, are compared with respect to their amino acid compositions, and their staining properties when the proteins are fixed on filter paper. The three types of basic proteins; somatic, arginine-rich spermatid histones, and protamine can be distinguished on the following basis. Somatic and testicular histones stain with fast green or bromphenol blue under the same conditions used for specific staining of histones in tissue preparations. The former histones lose most or all of their stainability after deamination or acetylation. Staining of the arginine-rich testicular histones remains relatively unaffected by this treatment. Protamines do not stain with fast green after treatment with hot trichloracetic acid, but are stained by bromphenol blue or eosin after treatment with picric acid. These methods provide a means for the characterization of nuclear basic proteins in situ. Their application to the early developmental stages of Helix aspersa show the following: After fertilization the protamine of the sperm is lost, and is replaced by faintly basic histones which differ from adult histones in their inability to bind fast green, and from protamines, by both their inability to bind eosin, and their weakly positive reaction with bromphenol blue. These "cleavage" histones are found in the male and female pronuclei, the early polar body chromosomes, and the nuclei of the cleaving egg and morula stages. During gastrulation, the histone complement reverts to a type as yet indistinguishable from that of adult somatic cells.     

Gomori's Hematoxylin as a cHromosome Stain

The chromic hematoxylin of Gomori (1941) can be used as an excellent chromosome stain after hydrolysis of the tissue in warm 1-N hydrochloric acid. The hydrolysis must be accurately timed for different material as in the case of the Feulgen reaction. The staining of sections can be performed at room temperature and requires about 15 minutes. For pieces of tissue and whole preparations, it is recommended to stain at 60°C. for 40 minutes. Sections stained at room temperature can be differentiated in 1% hydrochloric acid alcohol for one minute and can be counterstained with phloxine according to Gomori's formula. Whole preparations or sections stained at 60°C. must be differentiated in 45% acetic acid for half an hour or more. Tissue pieces may, after staining, be squashed and examined in the acetic acid, but the preparations can also be made permanent. The blue-black stain is very selective and has the advantage of giving high contrast, and it is nonfading, and insoluble in water and other common reagents. It proved definitely superior to other chromosome stains for difficult material such as planarians, rabbit blastocysts, and cleavage stages of sea urchins. Though both the procedure and the result of this method show some similarity to the Feulgen reaction nothing can be said with certainty about its chemical basis.     

Histone- and chromatin-binding activity of template activating factor-I

Template activating factor-I (TAF-I) is a histone-binding chromatin remodeling factor. We recently found that TAF-I is capable of mediating decondensation of Xenopus sperm chromatin by releasing sperm-specific basic proteins. Here we present evidence that TAF-I preferentially binds to histone H3 among four core histones. Immunofluorescent staining revealed that TAF-I binds to the decondensed sperm chromatin, of which protein components predominantly consist of histones H3 and H4.     

Histone H1 variants as sperm-specific nuclear proteins of Rana catesbeiana,and their role in maintaining a unique condensed state of sperm chromatin

Amino acid analyses of nuclear basic proteins of an anuran amphibian, Rana catesbeiana, revealed that they are comprised of a full set of core histones and three types of lysine-rich, sperm-specific proteins. On the basis of their amino-acid compositions and partial amino-acid sequences of their trypsin-resistant cores, the sperm-specific proteins could be defined as members of the histone H1 family. Both micrococcal nuclease digestion and electron microscopy indicated that sperm chromatin consists of nucleosomal and fibrillar DNA structures which are irregularly interspersed with each other. When sperm nuclei were incubated with nucleoplasmin, nuclei decondensed to some extent, and the sperm-specific H1s were removed, but not completely. The residual sperm-specific histone H1 variants were also found in reconstituted male pronuclear chromatin, comprising regularly spaced nucleosomes. We conclude that sperm-specific histone H1 variants are essential for chromatin condensation in the sperm nuclei, but that their complete removal is not necessary for the remodeling into somatic chromatin that takes place after fertilization. Mol. Reprod. Dev. 47:181–190, 1997. © 1997 Wiley-Liss, Inc.     

Neuron Staining by Basic Dyes Versus Basic Metal-Dye Complexes; Differences Shown by Histochemical Blocking Reactions

Various blocking procedures were applied to sections of paraffin-embedded, formalin-fixed cat spinal cord. Treated sections and untreated controls were stained with cresyl violet acetate or gallocyanine-chrome alum. Although both dyes have been said to stain by simple salt formation it was found that staining was affected differently for each dye by the blocking procedures, and also that staining of neuron nuclei differed in the controls. In these, the cresyl violet acetate stained only the nucleoli within the nucleoplasm whereas gallocyanine-chrome alum stained much more material of unknown composition and function. It is proposed that if cresyl violet acetate and other basic dyes stain by salt linkage, and can be specific for nucleic acid and other highly acid materials, then gallocyanine and other basic metal dye complexes can not be specific for nucleic acid and do not stain by a simple salt linkage.     

Arginine-rich nucleoprotein transition occurs in the two size classes of spermatozoa of Drosophila subobscura males

The normal male of Drosophila subobscura displays polymegaly, which is the presence of two sizes of spermatozoa in the same testis. It is still unknown whether both kinds of sperm are able to fertilize the egg. An indicator of normal functioning of Drosophila spermatozoa is the replacement of the somatic histones by sperm-specific arginine-rich nucleoproteins during spermiogenesis. The appearance of these arginine-rich nucleoproteins in the two kinds of sperm was investigated using the fluorescent dye sulfoflavine, which stains basic proteins at pH 8. In the spherical nuclei of early spermatids of Drosophila subobscura the somatic histones fluoresced strongly, but fluorescence could not be detected in later stages when the spermatid nuclei were elongating. After elongation, however, the nuclei of both kinds of sperm, long and short, fluoresced brightly again, due to the presence of sperm-specific arginine-rich nucleoproteins. Half of the cysts of both types contained spermatid nuclei with aberrant fluorescent pattern including 5–9% of both cyst types which do not undergo histone transition at all. These results indicate that both sperm types may be functional.     

Improved Histology for the Chick-Quail Chimera

A simple modification of nuclear staining after acid hydrolysis has been made which provides easy identification of quail nuclear markings in a chick-quail chimera. This method also improves the histologic detail normally seen with hematoxylin and eosin when compared to the more commonly used Feulgen reaction. Embryonic tissues can be fixed in Zenker's or Helly's solution and the sections obtained are hydrolyzed in acid (3.5 N HCl at 37 C for 40-50 min). After acid hydrolysis the sections are stained with hematoxylin and eosin rather than Schiff reagent and fast green. The interphase nuclei of chick cells show homogeneous or mottled purplish blue staining, while quail nuclei contain a dark blue spot. This staining corresponds to the reddish purple staining of the quail's heterochromatin seen adjacent to the nucleolus in the standard Feulgen stain. This new technique facilitates identification of quail cell types in the chick host and provides superior histology of the chick tissues by demonstrating cytoplasmic detail.     

Changes in accessibility of DNA to various fluorochromes during spermatogenesis

Accessibility of mouse testicular and vas deferens (vas) sperm cell DNA to acridine orange, propidium iodide, ellipticine, Hoechst 33342, mithramycin, chromomycin A3, 4'6-diamidino-2-phenylindole (DAPI), and 7-amino-actinomycin D (7-amino-AMD) was determined by flow cytometry. Permeabilized cells were either stained directly or after pretreatment with 0.06 N HCl. For histone-containing tetraploid, diploid, and round spermatid cells, HCl extraction of nuclear proteins caused an approximately sixfold increase of 7-amino-AMD stainability but had no significant effect on DAPI stainability. For these same cell types, the stainability with other intercalating (acridine orange, propidium iodide, ellipticine) and externally binding (Hoechst 33342, mithramycin, chromomycin A3) dyes was increased by 1.6- to 4.0-fold after HCl treatment. In sharp contrast, HCl treatment of vas sperm did not increase the staining level of 7-amino-AMD, DAPI, or propidium iodide but did increase the staining level for the other intercalating dyes (1.3- to 1.5-fold) and external dyes (1.3- to 1.9-fold). Elongated spermatids that contain a mixture of protein types including histones, transition proteins, and protamines demonstrated the greatest variability of staining with respect to type of stain and effect of acid extraction of proteins. In general, for nearly all dyes, the round spermatids had an increased level and tetraploid cells had a decreased level of stainability relative to the same unit DNA content of diploid cells. The observed differential staining is discussed in the context of chromatin alterations related to the unique events of meiosis and protein displacement and replacement during sperm differentiation.     

Nucleosomal organization of a part of chromatin in mollusc sperm nuclei with a mixed basic protein composition

The structural organization of mature sperm chromatin from three representatives of theMytilidae family has been studied. The acid-soluble proteins in these species nuclei are primarily sperm-specific (approximately 80%) with the remainder being core histones. Previously, we have shown that the mature sperm nuclei of these molluscs are compact, dense structures formed by interaction of the spermspecific proteins with DNA (1). Here we show that: a) although the histones are minor chromatin protein fraction, they still organize a part (20–25%) of the total DNA into nucleosomes; b) one of the sperm-specific proteins, different from somatic H1 or H5 histones participates in the formation of the beaded structures.     

SpermBlue®: A new universal stain for human and animal sperm which is also amenable to automated sperm morphology analysis

Abstract

Our study was aimed at exploring a simple procedure to stain differentially the acrosome, head, midpiece, and flagellum of human and animal sperm. A further prerequisite was that sperm morphology of the stained samples could be analyzed using automated sperm morphology analysis (ASMA). We developed a new staining process using SpermBlue® fixative and SpermBlue® stain, which are iso-osmotic in relation to semen. The entire fixation and staining processes requires only 25 min. Three main steps are required. First, a routine sperm smear is made by either using semen or sperm in a diluting medium. The smear is allowed to air dry at room temperature. Second, the smear is fixed for 10 min by either placing the slide with the dried smear in a staining tray containing SpermBlue® fixative or by adding 1 ml SpermBlue® fixative to the slide. Third, the fixed smear is stained for 15 min by either immersing the slide in a staining tray containing SpermBlue® stain or adding four drops of SpermBlue® stain to the fixed smear. The stained slide is dipped gently in distilled water followed by air drying and mounting in DPX® or an equivalent medium. The method is simple and suitable for field conditions. Sperm of human, three monkey species, horse, boar, bull, ram, mouse, rat, domestic chicken, fish, and invertebrate species were stained successfully using the SpermBlue® staining process. SpermBlue® stains human and animal sperm different hues or intensities of blue. It is possible to distinguish clearly the acrosome, sperm head, midpiece, principal piece of the tail, and even the short end piece. The Sperm Class Analyzer® ASMA system was used successfully to quantify sperm head and midpiece measurements automatically at either 600 × or 1000 × magnification for most of the species studied.     

A CHROMATOGRAPHIC STUDY OF HYDROLYSIS IN THE FEULGEN NUCLEAL REACTION

In a study on Feulgen hydrolysis of frozen-dried alcohol-fixed lily anthers, a chromatographic technique was developed to analyze the acid hydrolysate for some of the degradation products of nucleic acid. Hydrolysis was accomplished by 10 per cent perchloric acid at 20°C., and a typical hydrolysis time-Feulgen intensity curve was obtained, with maximum staining occurring at 19 hours. Microphotometric measurements indicated that the amount of stain per nucleus was no different from amount in nuclei fixed and hydrolyzed by more conventional procedures. Uracil-containing material (from ribonucleic acid) was almost completely separated from thymine-containing material (deoxyribonucleic acid) of tissue sections by acid treatment for 1½ hours. Adenine (purines), as the base, was effectively all removed from the deoxyribonucleic acid at the time of optimum hydrolysis. Detectable amounts of thymine-containing material appeared in the hydrolysate shortly after the onset of hydrolysis; and the amount increased rapidly with increased hydrolysis time. At the time of optimum hydrolysis approximately two-thirds of the total deoxyribonucleic acid thymine was lost. The removal of these thymine-containing fragments was linear with respect to time during the first 24 hours and occurred at a relatively high rate. Removal after 24 hours was also linear but was at a markedly lower rate. These results would suggest that two kinds of deoxyribonucleic acid exist in lily anthers; an acid-labile fraction amounting to approximately three-fourths of the total, and an acid-resistant fraction making up the remainder. In the Feulgen procedure much of the labile fraction is lost by the time of optimum hydrolysis and is not stained; most of the stable fraction remains in the tissue and is stained. In light of these findings the use of the Feulgen method as a means of determining cytochemically relative amounts of deoxyribonucleic acid in nuclei by measuring their Feulgen dye content was discussed.     

On the diversity of sperm histones in the vertebrates: IV. Cytochemical and amino acid analysis in Anura

The variability of sperm histones in frogs has been studied by cytochemical and amino acid analyses. Cytochemically, Rana sperm proteins fall into Bloch's ('69, '76) type 4 somatic-like histone category, while Xenopus and Bufo have type 3 intermediate sperm histones. Extractability in 5% trichloroacetic acid (TCA) at different temperatures splits this type 3 category into two groups: type 3B intermediate sperm histones of Bufo are extractable at 85-90 degrees C, while Xenopus intermediate type 3A sperm histones require temperatures of 95-100 degrees C for extraction. Amino acid analysis confirms that Rana sperm histones are of the nucleosomal type, with a testis-specific, very lysine-rich H1 histone. The sperm protein in Bufo is richer in arginine than the proteins in Xenopus. Both of these genera contain lysine and histidine as well as arginine in their sperm proteins. These results confirm earlier electrophoretic data (Kasinsky et al., '78) and indicate that sperm histones in the order Anura can vary markedly between different genera.     

Staining Mast Cells for Morphometric Evaluation on an Image Analysis System

Multiple skin sections from three nonhuman primates (Macaca mulatta) and three hairless guinea pigs (Cavia porcellus) were stained with 12 different histologic stains to determine whether mast cells could be selectively stained for morphometric analysis using an image analysis system (IAS). Sections were first evaluated with routine light microscopy for mast cell granule staining and the intensity of background staining. Methylene blue-basic fuchsin and Unna's method for mast cells (polychrome methylene blue with differentiation in glycerin-ether) stained mast cell granules more intensely than background in both species. Toluidine blue-stained sections in the guinea pig yielded similar results. Staining of the nuclei of dermal connective tissue was enhanced with the methylene blue-basic fuchsin and toluidine blue stains. These two stains, along with the Unna's stain, were further evaluated on an IAS with and without various interference filters (400.5-700.5 nm wavelengths). In both the methylene blue-basic fuchsin and toluidine blue stained sections, mast cell granules and other cell nuclei were detected together by the IAS. The use of interference filters with these two stains did not distinguish mast cell granules from stained nuclei. Unna's stain was the best of the 12 stains evaluated because mast cell granule staining was strong and background staining was faint. This contrast was further enhanced by interference filters (500.5-539.5 nm) and allowed morphometric measurements of mast cells to be taken on the IAS without background interference.     

Aldehyde Pararosanilin (True Aldehyde Fuchsin) Stains Purified Insulin, Oxidized or Not, Following Adequate Fixation with Either Formalin or Bouin'S Fluid

Gomori reported that aldehyde fuchsin stained the granules of pancreatic islet beta cells selectively and without need of permanganate pretreatment. Others adopted permanganate oxidation because it makes staining faster though much less selective. All aldehyde fuchsins are not equivalent, being made from “basic fuchsin” whose composition may vary from pure pararosanilin to one of its methylated homologs, rosanilin or a mixture. Mowry et al. have shown that only aldehyde fuchsin made from pararosanilin stained unoxidized pancreatic beta cells (PBC). Aldehyde fuchsins made from methylated homologs of pararosanilin stain PBC cells only after oxidation, which induces basophilia of other cells as well; these are less selective for PBC.

Is the staining of PBC by aldehyde fuchsins due to insulin? Others have been unable to stain pure insulin with aldehyde fuchsins except in polyacrylamide gels and only after oxidation with permanganate. They have concluded that insulin contributed to the staining of oxidized but not of unoxidized PBC. This view denies any inherent validity of the more selective staining of unoxidized PBC cells as an indication of their insulin content.

We describe here indisputable staining of unoxidized pure insulins by aldehyde fuchsin made with pararosanilin. Dried spots of insulin dissolved in the stain unless fixed beforehand. Spots of dried insulin solution made on various support media and fixed in warm formalin vapor were colored strongly by the stain. Insulin soaked Gelfoam® sponges were dried, fixed in formalin vapor and processed into paraffin. In unoxidized paraffin sections, presumed insulin inside gel spaces was stained strongly by aldehyde pararosanilin. Finally, the renal tubules of unoxidized paraffin sections of kidneys from insulin-injected mice fixed in either Bouin's fluid or formalin were loaded with material stained deeply by aldehyde pararosanilin. This material was absent in renal tubules of mice receiving no insulin. The material in the spaces of insulin-soaked gels and in the renal tubules of insulin-injected mice was proven to be insulin by specific immunostaining of duplicate sections. The same material was also stained by aldehyde pararosanilin used after permanganate. So, this dye stains oxidized or unoxidized insulin if fixed adequately.     

Aldehyde pararosanilin (true aldehyde fuchsin) stains purified insulin, oxidized or not, following adequate fixation with either formalin or Bouin's fluid

Gomori reported that aldehyde fuchsin stained the granules of pancreatic islet beta cells selectively and without need of permanganate pretreatment. Others adopted permanganate oxidation because it makes staining faster though much less selective. All aldehyde fuchsins are not equivalent, being made from "basic fuchsin" whose composition may vary from pure pararosanilin to one of its methylated homologs, rosanilin or a mixture. Mowry et al. have shown that only aldehyde fuchsin made from pararosanilin stained unoxidized pancreatic beta cells (PBC). Aldehyde fuchsins made from methylated homologs of pararosanilin stain PBC cells only after oxidation, which induces basophilia of other cells as well; these are less selective for PBC. Is the staining of PBC by aldehyde fuchsins due to insulin? Others have been unable to stain pure insulin with aldehyde fuchsins except in polyacrylamide gels and only after oxidation with permanganate. They have concluded that insulin contributed to the staining of oxidized but not of unoxidized PBC. This view denies any inherent validity of the more selective staining of unoxidized PBC cells as an indication of their insulin content. We describe here indisputable staining of unoxidized pure insulins by aldehyde fuchsin made with pararosanilin. Dried spots of insulin dissolved in the stain unless fixed beforehand. Spots of dried insulin solution made on various support media and fixed in warm formalin vapor were colored strongly by the stain. Insulin soaked Gelfoam sponges were dried, fixed in formalin vapor and processed into paraffin. In unoxidized paraffin sections, presumed insulin inside gel spaces was stained strongly by aldehyde pararosanilin. Finally, the renal tubules of unoxidized paraffin sections of kidneys from insulin-injected mice fixed in either Bouin's fluid or formalin were loaded with material stained deeply by aldehyde pararosanilin. This material was absent in renal tubules of mice receiving no insulin. The material in the spaces of insulin-soaked gels and in the renal tubules of insulin-injected mice was proven to be insulin by specific immunostaining of duplicate sections. The same material was also stained by aldehyde pararosanilin used after permanganate. So, this dye stains oxidized or unoxidized insulin if fixed adequately.     

Thionin Staining of Paraffin and Plastic Embedded Sections of Cartilage

The usefulness of thionin for staining cartilage sections embedded in glycol meth-acrylate (GMA) and the effect of decalcification on cartilage sections embedded in paraffin and GMA were assessed. Short decalcification periods using 5% formic acid or 10% EDTA did not influence the staining properties or the morphology of cartilage matrix and chondrocytes. The standard stain safranin O-fast green for differential staining of cartilage was used as control in these experiments. Prolonged exposure of safranin P stained sections to fast green resulted in disappearance of the safranin O stained matrix, thereby hampering the quantitative measurement of negatively charged glycosaminoglycans (GAG). Thionin stained evenly throughout all cartilage layers, independent of the staining times. In contrast to safranin 0, thionin did not show meta-chromasia in nondehydrated cartilage sections, which made it more suitable for assessing cartilage quality in GMA embedded cartilage. To evaluate the selectivity of thionin staining in cartilage, chondroitinase ABC and trypsin digestions were carried out. Thionin staining was prevented by these enzymes in the territorial matrix, representing the interlacunar network and the chondrocyte capsule. Staining with thionin of the interterritorial matrix was only slightly reduced, possibly representing keratan sulfate and hyaluronic acid in cartilage of elderly patients. Comparison of thionin stained GMA embedded cartilage with safranin O stained paraffin embedded sections showed significant similarity in optical densitometry, indicative of the specificity of thionin bound to negatively charged GAG in cartilage. In GMA embedded cartilage morphology was relatively intact compared to paraffin embedded sections due to less shrinkage of chondrocytes and the interlacunar network.