Effects of Hydrogen Ion Dissociation and Concentration on The Reactivity of Dichromate-Fixed Tissue Components to the Pas Procedure: Recommendations for Reducing Undesirable Background Staining

The undesirable PAS reactivity of cytoplasmic aldehydes after dichromate fixation can be suppressed without affecting selective staining by lowering the pH of Lillie's Cold Schiff reagent to 1.5. Alternatively, dilution of pH 2.2 Cold Schiff reagent with distilled water (1:2) is recommended. Hydrogen ion concentration and dissociation affect the rate of color formation in various PAS positive sites differentially with respect to the time of incubation in Schiff reagent. Based on these experiments, aldehydes exposed in different tissue components appear to be chemically distinct and separable depending on the rate of color formation and duration of incubation in Schiff reagent.     

Effects of hydrogen ion dissociation and concentration of the reactivity of dichromate-fixed tissue components to the PAS procedure: recommendations for reducing undesirable background staining.

The undesirable PAS reactivity of cytoplasmic aldehydes after dichromate fixation can be suppressed without affecting selective staining by lowering the pH of Lillie's Cold Schiff reagent to 1.5. Alternatively, dilution of pH 2.2 Cold Schiff reagent with distilled water (1:2) is recommended. Hydrogen ion concentration and dissociation effect the rate of color formation in various PAS positive sites differentially with respect to the time of incubation in Schiff reagent. Based on the experiments, aldehydes exposed in different tissue components appear to be chemically distinct and separable depending on the rate of color formation and duration of incubation in Schiff reagent.     

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.     

Histochemical identification of side chain substituted O-acylated sialic acids: the PAT-KOH-Bh-PAS and the PAPT-KOH-Bh-PAS procedures

Summary Two new methods, based on the original periodic acid-Thionin Schiff-saponification-periodic acid-Basic Fuchsin Schiff (PAT-KOH-PAS) technique of Cullinget al. (1976), have been devised for the histochemical identificantion of side-chainO-acylated sialic acids. In the first of these, the periodic acid-Thionin Schiff-saponification-borohydride reduction-periodic acid-Basic Fuchsin Schiff (PAT-KOH-Bh-PAS) procedure, the specificity of the original PAT-KOH-PAS technique was improved by: (a) extending, when necessary, the initial period of periodate oxidation, (b) increasing the period of exposure to Thionin Schiff reagent from 30 min to 4 h, (c) using a Thionin Schiff reagent prepared by a different method, (d) interposing a borohydride reduction step between the saponification and PAS steps and, (e) extending the period of oxidation in the final PAS step from 10 to 30 min. In the second procedure, the periodic acid-phenylhydrazine-Thionin Schiffborohydride reduction-periodic acid-Basic Fuchsin Schiff (PAPT-KOH-Bh-PAS), based on the periodic acid-phenylhydrazine-Schiff (PAPS) technique of Spicer (1961), blue Thionin Schiff staining was confined to sialic acid residues with oxidizable side chainvicinal diols by interposing a treatment with 0.5% (w/v) aqueous phenylhydrazine hydrochloride for 2 h at room temperature between the initial periodic acid oxidation and the Thionin Schiff steps of the PAT-KOH-Bh-PAS procedure. These procedures are discussed within the general framework of the methods available for the histochemical identification of side-chainO-acylated sialic acids.     

A Simple Procedure for Crystallization of the Schiff Reagent

Formation of crystals in Schiff reagents prepared from SO₂ gas previously has been reported either soon after preparation, using high dye concentrations and heating, or after long periods of storage at room temperature. With the first type of procedure only a low yield of crystals accompanied by dye precipitation was obtained. Crystallization without dye precipitation took place if the reagent, prepared with pararosaniline base or chloride in a saturated SO₂ solution, was stored for a sufficient time at room temperature in partly filled flasks. These crystals remained colorless if washed with acid alcohol after being separated by filtration. Schiff reagents layered with paraffin oil or supplemented with 0.1 M hydroquinone took much longer to crystallize, suggesting that crystallizaticn is promoted by the partial oxidation of sulfurous acid to sulfuric acid. A high yield of crystals can be obtained at room temperature after as little as 24 hr by adding 0.04 M of H₂SO₄ to a Schiff reagent prepared with 2% pararosaniline chloride in a saturated SO₂ solution. A Schiff reagent prepared with only 0.2% of these crystals gives an intense staining in the Feulgen and in the Periodic acid-Schiff reactions.     

The apparent failure of sodium borohydride reduction to block further PAS reactivity in rat epithelial mucins

Synopsis Sections of rat small intestine were oxidized with 1% periodic acid for periods of 1, 2, 5, 10 and 30 min and were subsequently either (a) stained with Schiff reagent, or (b) reduced with sodium borohydride and then treated with either Schiff reagent alone or or by the standard PAS procedure. It was found that whereas sodium borohydride reduction abolished all Schiff staining, initial periods of oxidation in excess of 10 min were necessary to abolish any subsequent PAS reactivity. The theoretical and practical significance of these data is discussed in relation to the recent publication of Bayliss & Adams (1976).     

Pararosaniline or acriflavine-Schiff staining of epoxy embedded tissue after periodic acid oxidation in ethanol: a method suitable for morphometric and fluorometric analysis of glycogen

A method for preparing tissue sections for automatic image analysis of glycogen is described. Large semithin sections of epoxy embedded tissue fixed in glutaraldehyde-osmium were stained with Schiff reagent and acriflavine (fluorescent staining) after resin removal and periodic acid oxidation in ethanol. We found it essential to avoid tissue rehydration before final staining. The Schiff stain permits an assessment of the cellular volume of glycogen, and the acriflavine allows a fluorometric evaluation of glycogen density.     

Evaluation of Preparation, Staining and Microscopic Techniques for Counting Incremental Lines in Cementum of Human Teeth

Apposition of cementum occurs in phases resulting in two types of layers with different optical and staining properties that can be observed by light microscopy. Narrow, dark staining incremental lines are separated by wider bands of pale staining cementum. The distance from one line to the next represents a yearly increment deposit of cementum in many mammals, and counting these lines has been used routinely to estimate the age of the animals. Incremental lines in cementum have also been observed in sections of human teeth, and the object of the present investigation was to examine a number of methods for preparing and staining them for counting. Longitudinal and transverse sections, either ground or decalcified, were cut from formalin fixed human dental roots, paraffin embedded or frozen, and stained using several techniques. The cementum was investigated using conventional light, fluorescence, polarized light, confocal laser scanning, interference contrast, phase contrast, and scanning electron microscopy. Incremental lines in the cementum could be observed in ground sections and, following decalcification, in both frozen and paraffin embedded sections. Toluidine blue, cresyl violet, hematoxylin, or periodic acid Schiff (PAS) stained incremental lines allowing differentiation by conventional light microscopy. Contrast was best using fluorescence microscopy and excitation by green light since the stained cemental bands, but not the incremental lines, fluoresced after staining with cresyl violet, PAS or hematoxylin and eosin. The results with other microscopic techniques were unsatisfactory. Since incremental lines are not destroyed by acids and stain differently than the remaining cementum, it is likely that they possess an organic structure which differs from the cementum. Incremental lines in human dental cementum could be observed best using decalcified sections stained with cresyl violet excited by green light.     

A new histochemical method for the selective periodate oxidation of total tissue sialic acids

Summary Evaluation of the intensity of the periodic acid—Schiff (PAS) staining produced following oxidation for 1 h at 4°C with 0.4mm periodic acid in approximately 1m hydrochloric acid indicated that this reagent completely oxidized all available sialic acid residues of either the sialo- or sialosulphoglycoproteins of human and rat colon or the sialoglycoproteins of rat sublingual gland. These conditions produced no visible Schiff staining of either neutral macromolecules orvicinal diols located on hexose, 6-deoxyhexose orN-acetylhexosamine residues (neutral sugars) of sialo- and sialosulphoglycoproteins. Furthermore, there was no extraction of epithelial glycoproteins or de-O-acylation of side chain substituted sialic acid residues. These data demonstrate that 0.4mm periodic acid in approximately 1m hydrochloric acid can be used as a specific reagent for the selective visualization of sialic acids in the PAS procedure.Studies of the mechanism of the oxidation of neutral sugars with 0.4mm periodic acid in approximately 1m hydrochloric acid indicated that their lack of PAS reactivity was not due to the production of Schiff unreactive hemiacetals or hemialdals. It is suggested that the selectivity of 0.4mm periodic acid in approximately 1m hydrochloric acid is a result of an increase in the rate of the oxidation of the sialic acid residues together with a decrease in the rate of oxidation of neutral sugars.     

A modified PAS stain combined with immunofluorescence for quantitative analyses of glycogen in muscle sections

Simultaneous analyses of glycogen in sections with other subcellular constituents within the same section will provide detailed information on glycogen deposition and the processes involved. To date, staining protocols for quantitative glycogen analyses together with immunofluorescence in the same section are lacking. We aimed to: (1) optimise PAS staining for combination with immunofluorescence, (2) perform quantitative glycogen analyses in tissue sections, (3) evaluate the effect of section thickness on PAS-derived data and (4) examine if semiquantitative glycogen data were convertible to genuine glycogen values. Conventional PAS was successfully modified for combined use with immunofluorescence. Transmitted light microscopic examination of glycogen was successfully followed by semiquantification of glycogen using microdensitometry. Semiquantitative data correlated perfectly with glycogen content measured biochemically in the same sample (r²=0.993, P<0.001). Using a calibration curve (r²=0.945, P<0.001) derived from a custom-made external standard with incremental glycogen content, we converted the semiquantitative data to genuine glycogen values. The converted semiquantitative data were comparable with the glycogen values assessed biochemically (P=0.786). In addition we showed that for valid comparison of glycogen content between sections, thickness should remain constant. In conclusion, the novel protocol permits the combined use of PAS with immunofluorescence and shows valid conversion of data obtained by microdensitometry to genuine glycogen data.     

Pararosaniline Or Acriflavine-Schiff Staining of Epoxy Embedded Tissue After Periodic Acid Oxidation in Ethanol: A Method Suitable for Morphometric and Fluorometric Analysis of Glycogen

A method for preparing tissue sections for automatic image analysis of glyco-gen is described. Large semithin seaions of epoxy embedded tissue fixed in glutaraldehyde osmium were stained with Schiff reagent and acriflavine (fluorescent staining) after resin removal and periodic acid oxidation in ethanol. We found it essential to avoid tissue rehydra-tion before final staining. The Schiff stain permits an assessment of the cellular volume of glycogen, and the acriflavine allows a fluorometric evaluation of glycogen density.     

THE PAS/ACCUMULATION BODIES IN PROROCENTRUM LIMA AND PROROCENTRUM MACULOSUM (DINOPHYCEAE) ARE DINOFLAGELLATE LYSOSOMES1

Numerous spherical bodies containing electron-dense material, fibrous material, and membranous material are present in the cytoplasm of two dinoflagellate species, Prorocentrum lima (Ehr.) Dodge and Prorocentrum maculosum Faust. Similar bodies have been observed in other dinoflagellates and have been termed accumulation bodies or PAS bodies. In both Prorocentrum species, these bodies autofluoresce under blue light excitation and increase in size with cell culture age. They possess acid phosphatase activity, react positively with the periodic acid/Schiff reagent, and stain with acridine orange. All these properties are characteristic of eukaryotic lysosomes; thus, we propose that dinoflagellate accumulation bodies and PAS bodies are identical organelles and are, in fact, dinoflagellate lysosomes.     

Periodic acid-Schiff reaction combined with quantitative autoradiography of 3H-thymidine- or 85S-sulfate-Labeled epithelial cells of colon

Summary The influence of periodic acid-Schiff staining on grain counts was examined using autoradiography of guinea pig colon labeled with either ³H-thymidine or ³⁵S-sulfate. Prestaining decreased the grain count, an effect due to the Schiff reagent but not to periodic acid. Poststaining altered the grains which were partly or completely lost, an effect due to periodic acid but not to the Schiff reagent. The suggested procedure is to pretreat the sections with periodic acid, process for autoradiography, and poststain with the Schiff reagent. No silver grain is then lost.     

Prestaining Oxidation by Acidified H2O2 for Revealing Schiff-Positive Sites in Epon-Embedded Sections

Reliable production and identification of Schiff-positive sites on glutaraldehyde-osmium fixed 0.5-1 μsm Epon sections is accomplished by preoxidation of sections with 10% H₂O₂ acidified with H₂SO₄ (HPSA) to pH 3.2 (Pool, C. R., Stain Techn., 44: 75-9, 1969). Light green as a counterstain is used. Steps in the procedure are: HPSA, 1-2 min at 25-30 C; washing; 1% light green 3-5 min; brief rinse; Schiff reagent 1-3 min; washing; drying; clearing in xylene and mounting in resin. The use of acidified H₂O₂ prevents the common occurrence of Schiff background staining in glutaraldehyde-fixed tissues and permits optimum penetration of staining solutions. Sections were attached to glass slides without adhesive and were processed in Coplin jars. Prior to drying, excess solutions should be drained and wiped away with lens tissue to prevent formation of precipitate on the sections.     

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.     

Effects of fuchsin variants in aldehyde fuchsin staining

Aldehyde fuchsin stains pancreatic B cell granules, hypophyseal basophils, goblet cell mucins, gastric chief cells, hyaline cartilage, and elastica. Neither the chemical structure of aldehyde fuchsin nor its staining mechanism is known. This study was undertaken to clarify the role of the fuchsin component of aldehyde fuchsin in its staining reaction. The major findings of this investigation include: 1) single N-methylation of the fuchsin molecule abolishes staining of unoxidized pancreatic B cells, although it does not prevent reaction of fuchsin with paraldehyde; 2) aldehyde fuchsin is probably a Schiff base condensation product of pararosaniline and acetaldehyde; 3) a Schiff base structure alone cannot account for aldehyde fuchsin staining of unoxidized pancreatic B cells; 4) a fully potent aldehyde fuchsin is possibly a Tris-Schiff base derivative of pararosaniline.     

Histological and Histochemical Uses of Periodic Acid

Periodic acid acts upon the 1,2 glycol linkage (-CHOH -CHOH-) of carbohydrates in tissue sections to produce aldehyde (RCHO+RCHO) which can be colored with Schiff s reagent. The method can be used on frozen or paraffin sections and is useful as a reaction for carbohydrates of tissues: glycogen (in paraffin section only), mucin, basement membrane, reticulin, the colloid of the pituitary stalk and thyroid, some of the acidophile cells of the human anterior hypophysis, the granular cells of the renal arteriole, etc.

In abnormal tissues, it colors many of the “hyaline” materials— amyloid infiltrations, arteriolosclerotic hyaline, colloid droplets, mitotic figures, etc.

The histochemical uses of the periodic-acid-Schiff's reagent (PAS) need careful control because of the possibility of attachment of iodate or periodate to tissue constitutents, producing a recoloration of the Schiff's reagent. Whenever possible the positive reacting material should be further identified by other methods since Lison showed other substances besides aldehydes can recolorize SchifFs reagent.     

Hexazonium pararosaniline as a fixative for animal tissues

Hexazonium pararosaniline is a valuable reagent that has been used in enzyme activity histochemistry for 50 years. It is an aqueous solution containing the tris-diazonium ion derived from pararosaniline, an aminotriarylmethane dye, and it contains an excess of nitrous acid that was not consumed in the diazotization reaction. Other investigators have found that immersion for 2 min in an acidic (pH 3.5) 0.0015 M hexazonium pararosaniline solution can protect cryostat sections of unfixed animal tissues from the deleterious effects of aqueous reagents such as buffered solutions used in immunohistochemistry, while preserving specific affinities for antibodies. In the present investigation hexazonium pararosaniline protected lymphoid tissue and striated muscle against the damaging effects of water or saline. The same protection was conferred on unfixed sections treated with dilute nitrous or hydrochloric acid in concentrations similar to those in hexazonium pararosaniline solutions. Model tissues (solutions, gels or films containing gelatin and/or bovine albumin) responded predictably to well known cross-linking (formaldehyde) or coagulant (mercuric chloride) fixatives. Hexazonium pararosaniline solutions prevented the dissolution of protein gels in water only after 9 or more days of contact, during which time considerable swelling occurred. It is concluded that there is no evidence for a “fixative” action of hexazonium pararosaniline. The protective effect on frozen sections of unfixed tissue is attributable probably to the low pH of the solution.     

Hexazonium pararosaniline as a fixative for animal tissues

Hexazonium pararosaniline is a valuable reagent that has been used in enzyme activity histochemistry for 50 years. It is an aqueous solution containing the tris-diazonium ion derived from pararosaniline, an aminotriarylmethane dye, and it contains an excess of nitrous acid that was not consumed in the diazotization reaction. Other investigators have found that immersion for 2 min in an acidic (pH 3.5) 0.0015 M hexazonium pararosaniline solution can protect cryostat sections of unfixed animal tissues from the deleterious effects of aqueous reagents such as buffered solutions used in immunohistochemistry, while preserving specific affinities for antibodies. In the present investigation hexazonium pararosaniline protected lymphoid tissue and striated muscle against the damaging effects of water or saline. The same protection was conferred on unfixed sections treated with dilute nitrous or hydrochloric acid in concentrations similar to those in hexazonium pararosaniline solutions. Model tissues (solutions, gels or films containing gelatin and/or bovine albumin) responded predictably to well known cross-linking (formaldehyde) or coagulant (mercuric chloride) fixatives. Hexazonium pararosaniline solutions prevented the dissolution of protein gels in water only after 9 or more days of contact, during which time considerable swelling occurred. It is concluded that there is no evidence for a “fixative” action of hexazonium pararosaniline. The protective effect on frozen sections of unfixed tissue is attributable probably to the low pH of the solution.     

Hexazonium pararosaniline as a fixative for animal tissues.

Hexazonium pararosaniline is a valuable reagent that has been used in enzyme activity histochemistry for 50 years. It is an aqueous solution containing the tris-diazonium ion derived from pararosaniline, an aminotriarylmethane dye, and it contains an excess of nitrous acid that was not consumed in the diazotization reaction. Other investigators have found that immersion for 2 min in an acidic (pH 3.5) 0.0015 M hexazonium pararosaniline solution can protect cryostat sections of unfixed animal tissues from the deleterious effects of aqueous reagents such as buffered solutions used in immunohistochemistry, while preserving specific affinities for antibodies. In the present investigation hexazonium pararosaniline protected lymphoid tissue and striated muscle against the damaging effects of water or saline. The same protection was conferred on unfixed sections treated with dilute nitrous or hydrochloric acid in concentrations similar to those in hexazonium pararosaniline solutions. Model tissues (solutions, gels or films containing gelatin and/or bovine albumin) responded predictably to well known cross-linking (formaldehyde) or coagulant (mercuric chloride) fixatives. Hexazonium pararosaniline solutions prevented the dissolution of protein gels in water only after 9 or more days of contact, during which time considerable swelling occurred. It is concluded that there is no evidence for a "fixative" action of hexazonium pararosaniline. The protective effect on frozen sections of unfixed tissue is attributable probably to the low pH of the solution.