The Lipids Stained by the Periodic Acid-Schiff Technic

Unsaturated periodic acid-Schiff (PAS) stainable lipids of renal basement membranes are soluble in lipid solvents and do not add to the PAS staining in paraffin embedded sections. These lipids contribute to the staining of basement membranes in frozen sections. Pure sphingomyelin is stained by the PAS method if the oxidising solution is sufficiently acid and the time allowed for periodic oxidation is sufficient. This staining is considered to depend on splitting of the amide link between sphingosine and the fatty acid which leaves the 1-amino-2-hydroxyl grouping of sphingosine available for reacting with the periodic acid.     

An improved periodic acid-thiosemicarbazide-osmium technique to reveal glycoconjugates at the molecular level in situ

The periodic acid-thiocarbohydrazide or thiosemicarbazide-OsO4 method (Seligman AM, Hanker JS, Wasserkrug H, Katzoff L: J Histochem Cytochem 13:629, 1965) has been modified in order to obtain a periodic acid-Schiff (PAS)-like reaction for electron microscopy capable of visualizing structures at the molecular level in situ. Thiocarbohydrazide (TCH) and thiosemicarbazide (TSC) have been used dissolved in distilled water and bubbled with SO2. Treatment of previously oxidized thin sections with TCH (SO2) or TSC (SO2), followed by osmification, resulted in selective and very good staining of all the PAS-positive structures examined: glycogen, intestinal mucopolysaccharides, plasma membrane glycoproteins, basement membranes, Golgi apparatus, and collagen. The staining reaction was highly specific when TSC was used on thin sections from paraformaldehyde-fixed samples. The non-particulate end-reaction product made possible visualization of a periodic distribution of sugar residues in the 64-nm unit of collagen and the structural organization of the PAS-positive glycoconjugate components in the glomerular basement membrane.     

SILVER IMPREGNATION OF ULTRATHIN SECTIONS FOR ELECTRON MICROSCOPY

A new procedure is described for silver impregnation of thin sections for electron microscopy. Sections of various tissues, fixed in OsO₄ and embedded in methacrylate, were treated with an ammoniacal silver solution, directly or after oxidation with periodic acid or hydrogen peroxide. After OsO₄ fixation all cellular membranous systems exhibit a primary argentaffinity probably due to the reduction of ammoniacal silver solution by the reduced osmium bound to unsaturated lipids. Bleaching the sections with hydrogen peroxide removes the argentaffinity of protoplasmic structures. Treatment of the sections with periodic acid results in decreased argentaffinity of protoplasmic components while the argentaffinity of metaplasmic structures is greatly enhanced. The latter procedure appears particularly useful for enhancing the contrast of basement membranes.     

Demonstration of chondroitin sulphate and glycoproteins in articular cartilage matrix using periodic acid-Schiff (PAS) method

Summary Staining of articular cartilage by the periodic acid-Schiff (PAS) method was measured using microspectrophotometry. Standard PAS technique with 2 h oxidation produced a distinct Schiff reaction in the cartilage sections. The staining increased with depth of the articular cartilage demonstrating distribution of the glycoproteins. The modified PAS method included a second, longer periodic acid treatment, which made the uronic acid of glycosaminoglycans PAS-positive. The modified PAS method proved to be highly specific for chondroitin sulphate, which was determined from the samples with gas chromatography. A statistically significant correlation between the Schiff reactivity and galactosamine content of the sections was observed. It is concluded that for articular cartilage standard and modified PAS methods are useful procedures for demonstrating local changes of glycoproteins and chondroitin sulphate, respectively.     

Histochemical observations on Pneumocystis carinii: Selective demonstration of honeycomb forms

Summary Histochemical investigations of pulmonary lesions indicated selective coloration of membranes of honeycomb stages of Pneumocystis carinii by the periodic acid — sodium bisulfite — resorcin-fuchsin reaction for basement membranes; mucus, fibrin and other deposits in respiratory pathways did not react. These membranes were colored selectively also by the picro-Sirius Red F3BA method for collagens; fungi in tissues from patients with candidiasis remained unstained. For simultaneous demonstration of honeycomb and cyst forms of Pneumocystis carinii, sections were prestained with Grocott's modification of Gomori's methenamine-silver nitrate technic and then treated with the periodic acid-Schiff (PAS) or picro-Sirius Red F3BA reaction. In contrast to other Gram-positive microorganisms, cysts of Pneumocystis carinii were immediately decolorized by acetone-ether mixtures; this indicates differences in the mode of dye binding. Frequently, only one stage of Pneumocystis carinii was found in a given area. Hence a combination of reactions showing different stages is recommended for studies of small tissue samples.     

Ss blood Group Associated PAS-staining polymorphism of glycoprotein 3 from human erythrocyte membranes

Summary It is shown by discontinuous sodium dodecylsulfate (SDS) polyacrylamide gel electrophoresis of human red cell membranes, followed by periodic acid Schiff (PAS) staining and densitometry, that the band PAS-3 (monomeric Ss glycoprotein) exhibits a polymorphism with respect to its staining intensity. In membranes of the genotype SS the staining intensity of this band is about 1.5 times higher than in ss membranes. The experimental error of the method does not permit one to decide, whether membranes of blood type Ss exhibit an intermediate staining intensity or not. The SDS electrophoretic molecular weight of PAS-3 is the same for SS, Ss, or ss membranes. The ratio of PAS to coomassie blue staining intensities, which are a measure of sialic acid and protein content, respectively, are equal for PAS-3 in glycoprotein preparations from SS and ss erythrocytes. These data indicate that the above phenomenon reflects a difference in the glycoprotein content between SS and ss membranes.     

Luxol Fast Blue Combined With the Periodic Acid-Schiff Procedure for Cytological Staining of Kidney

Rat kidneys fixed in Regaud's fluid were stained by luxol fast blue (LFB), by the periodic acid-Schiff (PAS) method, and by LFB combined with PAS. When used separately the PAS stains the brush border, hyaline droplets and basement membranes reddish, the LFB stains the mitochondria, hyaline bodies and basement membranes greenish-blue. The combined LFB-PAS method stains the brush border reddish and the mitochondria dark blue, while the hyaline bodies and basement membranes are purplish colored. The LFB-PAS method provides color contrasts which show cytological features that are particularly significant in the kidney.     

Histochemical observations on Pneumocystis carinii: selective demonstration of honeycomb forms.

Histochemical investigations of pulmonary lesions indicated selective coloration of membranes of honeycomb stages of Pneumocystis carinii by the periodic acid--sodium bisulfite--resorcin-fuchsin reaction for basement membranes; mucus, fibrin and other deposits in respiratory pathways did not react. These membranes were colored selectively also by the picro-Sirius Red F3BA method for collagens; fungi in tissues from patients with candidiasis remained unstained. For simultaneous demonstration of honeycomb and cyst forms of Pneumocystis carinii, sections were prestained with Grocott's modification of Gomori's methenamine-silver nitrate technic and then treated with the periodic acid-Schiff (PAS) or picro-Sirius Red F3BA reaction. In contrast to other Gram-positive microorganisms, cysts of Pneumocystis carinii were immediately decolorized by acetone-ether mixtures; this indicates differences in the mode of dye binding. Frequently, only one stage of Pneumocystis carinii was found in a given area. Hence a combination of reactions showing different stages is recommended for studies of small tissue samples.     

Periodic acid incubation can replace hydrochloric acid hydrolysis and trypsin digestion in immunogold-silver staining of bromodeoxyuridine incorporation in plastic sections and allows the PAS reaction

Summary We have examined the possibility of improving the present methods of detecting bromodeoxyuridine (BrdU) and for combining the PAS reaction with the BrdU detection by means of immunogold-silver staining (IGSS). This was done in testes fixed in Carnoy or Bouin, and in parts of the small intestine which were fixed in Carnoy or periodate-lysine-paraformaldehyde (PLP). All tissues were embedded in a mixture of glycol methacrylate and butanediol-monocrylate. It was found to be impossible to carry out BrdU detection using HCl hydrolysis and trypsin digestion in combination with a PAS reaction. However, incubation of the plastic sections in periodic acid for a period of 30 minutes appeared to make it possible to eliminate the HCl denaturation step and to carry out a specific PAS reaction. Moreover, after incubation in periodic acid, trypsin digestion was no longer required to make the BrdU label accessible in GMA-embedded sections, nor to re-expose the antigenic sites in plastic sections of tissues fixed with cross-linking fixatives. In this way the loss of cell structures, which is inevitable when trypsin is used, can be avoided. Now a BrdU detection with improved morphology can be combined with the PAS reaction in the same plastic section in order to stain tissue carbohydrates. This is important for tumour diagnosis, where the PAS reaction can be very useful.     

Positive periodic acid-Schiff staining of acid mucopolysaccharides

Synopsis Evidence is presented to demonstrate that acid mucopolysaccharides will stain with the periodic acid-Schiff technique following prolonged oxidation with periodic acid. Smears of purified acid mucopolysaccharides begin to stain slightly with the Schiff reagent after 4 hr of periodate oxidation and reach an optimal staining intensity some time between 7 and 16 hr. The acid mucopolysaccharides in umbilical cord and cock's comb sections begin to stain at about 7 hr, reaching an optimum between 16 and 24 hr. It is suggested that the mechanism of staining of acid mucopolysaccharides in the PAS technique appears to derive from cleavage oftrans groups of the hexuronic acid fraction which require prolonged oxidation since the theoretical more yield is reached with difficulty. Moreover, it would appear that the mechanism of staining with glycogen under the usual conditions of PAS staining perhaps derives from end-group oxidation and subsequent Schiff staining of the engendered aldehydes since glycogen is almost entirely in thetrans configuration. Acid mucopolysaccharides, on the other hand, will not stain uncer the usual conditions as they appear to have a negligible proportion of end groups.     

A Selective Stain for Renal Basement Membranes

Paraffin sections from human kidneys fixed in Carnoy's fluid No. 2 were treated consecutively with periodic acid-sodium bisulfite and stained with resorcin-fuchsin. Basement membranes were colored black in cross sections, dark gray in tangential sections. Cytoplasm, nuclei, reticulin and collagen fibers remained unstained or were only lightly colored, depending on duration of fixation. Elastic fibers were colored black. In sections counterstained with Kernechtrot, the sharp black coloration of basement membranes and the pink staining of nuclei facilitated the study of glomerular lesions. After counterstaining with Van Gieson's picro-fuchsin, the black basement membranes contrasted well with the red reticulin and collagen fibers. Because this method does not require differentiation, it gave uniform results in the hands of different users. No fading was observed in section stored for 3 yr.     

A Cytochemical Technique for Demonstration of Lipids,Polysaccharides and Protein Bodies in Thick Resin Sections

An effective cytochemical technique for the simultaneous demonstration of lipids, polysaccharides and protein bodies in the same section from the tissue embedded in Epon 812 is described. Thick sections of peanut cotyledon are used for a typical sample according to the following procelures. Firstly, PAS reaction: (1) Oxidize sections in 0.5% periodic acid in 0.3% nitric acid for 10 min, (2) Wash in running water for 1–2 min and then pass through distilled water, (3) Stain in Schiff's reagent for 30 min, (4) Wash in sodium metabisulfite 3 times, 2 min for each time, (5) Wash in running water for 5 min and then pass through distilled water. Secondly, Sudan black B staining: (1) Rinse section in 70% ethanol for 1-2 min, (2) Stain in fresh 1% Sudan black B in 70% ethanol for 30–60 min at 40–60℃, (3)Rinse in 70% ethanol for 1 min and then in distilled water. Thirdly, Coomassie brilliant blue R staining: (1) Rinse sections in 7% acetic acid for 1–2 min, (2) Stain in I% Coomassie brilliant blue R in 7% acetic acid for 20 min at 60℃, (3) Differentiate in 0.1% acetic acid for I min, (4) Rinse in lunning water for 5 min and then pass through distilled water, (5) Dry at room temperature or in oven, 40℃. The dry sections mount in glycerin-gelatin. After the above three step staining, the three main compounds of the cell can be stained simultaneously. Starch grains and cellulose cell wall take cherry red colour, lipids appear in black, protein bodies are blue. The sealed slides can be kept permanently.     

Application of Phase-Contrast Microscopy to Schiff-Positive Material

It was observed that ground substance between the smooth muscle fibers in cerebral arteries stained by periodic acid-Schiff (PAS) was red as seen by ordinary bright-field microscopy (BF), but blue as observed by phase-contrast microscopy (PC). The basement membranes in the small intestine and around the kidney tubules, as well as the striated borders of the intestinal epithelium and the brush borders of the kidney tubules, were seen in blue when stained by PAS and observed by PC. The cytoplasm of PAS stained liver cells, when observed by PC, had irregular shaped areas of blue interspersed between the red material. This blue color was seen by PC after PAS, ninhydrin-Schiff and the Feulgen procedures. Our evidence suggests that this phenomena is characteristic of Schiff-positive material. Digestion by various enzymes: malt diastase, testicular hyaluronidase, collagenase, pepsin, pectinase, trypsin and DNase showed different effects on ground substance, liver cells, basement membranes, and brush and striated borders.     

Rapid and simple single nanogram detection of glycoproteins in polyacrylamide gels and on electroblots

The fluorescent hydrazide, Pro-Q Emerald 300 dye, may be conjugated to glycoproteins by a periodic acid Schiff's (PAS) mechanism. The glycols present in glycoproteins are initially oxidized to aldehydes using periodic acid. The dye then reacts with the aldehydes to generate a highly fluorescent conjugate. Reduction with sodium metabisulfite or sodium borohydride is not required to stabilize the conjugate. Though glycoprotein detection may be performed on transfer membranes, direct detection in gels avoids electroblotting and glycoproteins may be visualized within 2-4 h of electrophoresis. This is substantially more rapid than PAS labeling with digoxigenin hydrazide followed by detection with an antidigoxigenin antibody conjugate of alkaline phosphatase, or PAS labeling with biotin hydrazide followed by detection with horseradish peroxidase or alkaline phosphatase conjugates of streptavidin, which require more than eight hours to complete. Pro-Q Emerald 300 dye-labeled gels and blots may be poststained with SYPRO Ruby dye, allowing sequential two-color detection of glycosylated and nonglycosylated proteins. Both fluorophores are excited with mid-range UV illumination. Pro-Q Emerald 300 dye maximally emits at 530 nm (green) while SYPRO Ruby dye maximally emits at 610 nm (red). As little as 300 pg of alpha 1-acid glycoprotein (40% carbohydrate) and 1 ng of glucose oxidase (12% carbohydrate) or avidin (7% carbohydrate) are detectable in gels after staining with Pro-Q Emerald 300 dye. Besides glycoproteins, as little as 2-4 ng of lipopolysaccharide is detectable in gels using Pro-Q Emerald 300 dye while 250-1000 ng is required for detection with conventional silver staining. Detection of glycoproteins may be achieved in sodium dodecyl sulfate-polyacrylamide gels, two-dimensional gels and on polyvinylidene difluoride membranes.     

Histochemical Detection of Carbohydrates of Blastocystis hominis

The carbohydrates of Blastocystis hominis were detected by histochemical techniques using light and electron microscopy. B. hominis, fixed with various fixatives, followed by treatment with detergents, were stained with periodic acid-Schiff (PAS) or alcian blue (AB). Intense PAS reactions were observed in cells fixed with glutaraldehyde or 1/2 Karnovsky fixative. The cells fixed with other fixatives showed weak or no reactions with PAS staining. Similar results were seen in the case of AB stain. These results indicated that, depending on the fixative used, B. hominis contained PAS- or AB-reactive carbohydrates. At the electron microscopic level, ultrathin sections of B. hominis were stained with periodic acid methenamine silver (PA-MS) or periodic acid thiocarbohydrazide-silver proteinate (PA-TCH-SP) staining techniques. Intense, positive reactions with PA-MS or PA-TCH-SP were observed on the central vacuole, Golgi apparatus, and cytoplasmic vesicles. The filamentous layer showed moderate reactions with PA-MS, whereas in PA-TCH-SP stain, it was stained more densely. The staining intensity of the central vacuole varied from cell to cell. The presence of membrane fusions of the cytoplasmic vesicles with the central vacuole indicated the accumulation of carbohydrates in the central vacuole.     

Effect of brown spider venom on basement membrane structures

Loxoscelism or necrotic arachnidism are terms used to describe lesions and reactions induced by bites (envenomation) from spiders of the genus Loxosceles. Envenomation has been reported to provoke dermonecrosis and haemorrhage at the bite site and haemolysis, disseminated intravascular coagulation and renal failure. The purpose of this work was to study the effect of the venom of the brown spider Loxosceles intermedia on basement membrane structures and on its major constituent molecules. Light microscopy observations showed that L. intermedia venom obtained through electric shock, which reproduces two major signals of Loxoscelism in the laboratory, exhibits activity toward basement membrane structures in mouse Engelbreth-Holm-Swarm (EHS) sarcoma. Basement degradation was seen by a reduced periodic acid-Schiff (PAS) and alcian blue staining as well as by a reduced immunostaining for laminin when compared to control experiments. Electron microscopy studies confirmed the above results, showing the action of the venom on EHS-basement membranes and demonstrating that these tissue structures are susceptible to the venom. Using purified components of the basement membrane, we determined through SDS-PAGE and agarose gel that the venom is not active toward laminin or type IV collagen, but is capable of cleaving entactin and endothelial heparan sulphate proteoglycan. In addition, when EHS tissue was incubated with venom we detected a release of laminin into the supernatant, corroborating the occurrence of some basement membrane disruption. The venom-degrading effect on entactin was blocked by 1,10-phenanthroline, but not by other protease inhibitors such as PMSF, NEM or pepstatin-A. By using light microscopy associated with PAS staining we were able to identify that 1,10-phenanthroline also inhibits EHS-basement membrane disruption evoked by venom, corroborating that a metalloprotease of venom is involved in these effects. Degradation of these extracellular matrix molecules and the observed susceptibility of the basement membrane could lead to loss of vessel and glomerular integrity, resulting in haemorrhage and renal problems after envenomation.     

Immunohistochemical identification of tubular segments in percutaneous renal biopsies

Summary To identify the renal cortical tubular segments involved in tubulo-interstitial disease in formalin-fixed, paraffin-embedded percutaneous kidney biopsies, we developed multiple immunolabeling protocols using segment-specific tubular markers. The present study of biopsies from patients with minimal change or thin basement membrane nephropathy provides a baseline for interpretation of histopathology. Proximal tubules were stained either by the PAS reaction or by the biotinylated Phaseolus vulgaris erythroagglutinin (PHA-E)-streptavidin-gold-silver system (brush borders black). The anti-Tamm-Horsfall (THP) antibody-immunoperoxidase (aminoethylcarbazole, AEC-IPO), and anti-epidermal cytokeratins (ECK) antibodies-immunoalkaline-Fast Blue BB methods marked the distal straight tubules and the cortical collecting system red-brown and blue, respectively. When these immunolabelings were combined, the coapplication of AEC-PO-labeled peanut agglutinin (PNA) or anti-epithelial membrane antigen antibody-AEC-IPO technique (both are markers for distal nephron) visualized the apical membranes of distal convoluted tubules. In the protocol PHA-E + PNA + THP + ECK, the tubular basement membranes were outlined by the anti-laminin antibody-AEC-IPO staining, carried out simultaneously. The protocol PNA + THP + ECK + PAS was found to be a quite appropriate multiple immunolabeling method for the tubules, and is recommended for use as a tool in the study of tubulo-interstitial diseases.Abbreviations PAS periodic acid-Schiff reaction - PHAE Phaseolus vulgaris erythroagglutinin - PNA Peanut agglutinin - EMA epithelial membrane antigen - THP Tamm-Horsfall glycoprotein - ECK epidermal cytokeratins - PO peroxidase - Biot-PHA-E biotinylated PHA-E - APAAP complexes of alkaline phosphatase and mouse monoclonal anti-alkaline phosphatase - SWARI swine anti-rabbit immunoglobulins - FCS fetal calf serum - TBS Tris-buffered saline - AEC aminoethylcarbazole - DAB diaminobenzidine - FBBB Fast Blue BB - IA immunoalkaline - GL glomerulus - PT proximal tubule - DST distal straight tubule - DCT distal convoluted tubule - CCS cortical collecting system - CT connecting tubule - CD collecting duct     

Monoclonal antibody analysis of ocular basement membranes during development

As a first step in a study of the role(s) of basement membranes in ocular morphogenesis, we have produced a variety of monoclonal antibodies against native lens capsule from adult chicks, and have used these reagents to stain histological sections of ocular tissues from 4 1/2- to 18-day-old chicken embryos. Four different patterns of immunofluorescence were observed in sections of corneas of 18-day-old chicken embryos stained with these antibodies. The antibodies in group 1 stained the basement membranes of both the corneal epithelium and the endothelium (as well as Descemet's membrane). Those in groups 2 and 3 stained only the epithelial or endothelial basement membranes, respectively. The group 4 antibody stained the corneal stroma as well as Bowman's membrane and Descemet's membrane. The antibodies in group 1 could be further subdivided into groups 1a and 1b on the basis of temporal differences in the onset of staining in corneas from 4 1/2- to 7-day-old embryos. Thus, this series of monoclonal antibodies appears to recognize at least five different antigenic determinants. When these antibodies were used to stain sections of eyes at different stages of development, we found that the characteristic differential staining of some basement membranes was maintained throughout development, while the staining properties of others changed. This indicates that many of the ocular basement membranes may differ from one another in composition or conformation, and that at least some of them may undergo developmental changes. We also noticed a similarity in the pattern of fluorescence associated with the basement membranes of the limbal blood vessels and the corneal endothelium that is consistent with the hypothesis that the corneal endothelium is derived from the early periocular vascular endothelium. Our observations of developing corneas also revealed that the antigen recognized by the group 4 antibody may be produced by both the corneal epithelium and the stromal fibroblasts. The suitability of monoclonal antibodies for probing basement membrane heterogeneity is discussed.     

Immunohistochemistry combined with periodic acid-Schiff for bovine mammary gland with protothecal mastitis

Immunohistochemistry (IHC) of bovine cytokeratin combined with periodic acid-Schiff (PAS) was applied to study the pathogenesis, localization and distribution of Prototheca zopfii in bovine mammary protothecosis. The standard immunohistochemical procedure using anti-bovine cytokeratin was employed before and after PAS staining to optimize this combined method. The best results were obtained when IHC procedures were performed first. Most of the epithelial cells reacted strongly with the pancytokeratin antibody. Protothecal cell walls stained well with PAS. Algal organisms were present within the lumen and between the epithelial lining and basement membrane of the affected alveoli, but not inside the positive mammary epithelial cells. This combined staining method resulted in clear alveolar epithelial detail and good contrast between the epithelial cells and algae, and contributed to studying the pathogenesis of P. zopfii in mammary protothecosis.     

Glycogen in pancreatic islets of steroid diabetic rats

Summary In the islets of the rat pancreas, steroid diabetes induced by triamcinolon-acetonid leads to degranulation of the B cells and glycogen infiltration. The glycogen cannot be satisfactorily detected using methods like the chromic acid technique according to Bauer, staining with Best's carmine, or the usually applied periodic acid-Schiff (PAS) reaction. Glycogen detection is improved, however, when lead tetraacetate is used in place of periodic acid as oxidizing agent. When combining the carbohydrate detection method with the peroxidase — antiperoxidase (PAP) method used for immunocytochemical detection of the various pancreatic islet hormones, paraffin sections reveal that glycogen is primarily localized in granulated B cells; the degranulated B cells also contain glycogen, though in smaller amounts. In contrast, the islet cells containing somatostatin, glucagon and pancreatic polypeptide are nearly free of glycogen.This study was supported by the Deutsche Forschungsgemeinschaft K1 426/2