Philip Pizzolato

MAYER'S TANNIC ACID-FERRIC CHLORIDE STAIN FOR MUCINS'

Tannic acid in aqueous solution is bound to mucins in formalin-fixed and formalin-free fixed tissues and its presence can be detected with ferric chloride as a dark gray, blue-black to black complex. This colored compound is readily extracted by acids and some chelating and bleaching agents and is changed to a reddish brown by alkalis. Hydrolysis in 1.2 N hydrochloric acid at 60°C for 4 hr or hot trichloroacetic acid prevents the tannin-iron reaction. Acetyl chloride or bromide is able to inhibit the binding of tannic acid to the mucosubstances and saponification restores the characteristic reaction. Several mechanisms for the attachment of tannic acid to the mucins appear possible.

Hematoxylin Substitutes: A Survey of Mordant Dyes Tested and Consideration of the Relation of Their Structure to Performance as Nuclear Stains

In the search for hematoxylin substitutes 26 dyes were more or less extensively tested for performance as nuclear stains, usually in combination with aluminum, chronic, ferrous and ferric salts. Reports from the literature on hematoxylin substitutes were also considered, and efforts were made to obtain samples of favorably reported dyes and test them. The reports on anthocyanins include isolated reports on several berry juices and a considerable number of studies on Sambucus niger and Vaccinium myrtillus. None of these have so far been tested by us. Otherwise favorable reports have appeared on eleven synthetic dyes and on carmine, brazilin, and hematin. Except for one of the synthetics, naphthazarin, which is no longer fractured, we had samples of all of these. In addition, more or less unsuccessful trials were made on twelve dyestuffs, some of which were new syntheses designed to combine chelating capacity with nucleophilia. Following Fygs report of blue nuclear staining with chrome alum carmine, trial was made to change the red nuclear stain of kernechtrot by altering the metal mordant. The most successful dyes were phenocyanin TC, gallein, fluorone black, alizarin cyanin BB and alizarin blue S. Celestin blue B with an iron mordant is quite successful if properly handled to prevent gelling of solutions.

HISTOCHEMICAL AZO COUPLING REACTIONS A CATECHOLAMINE IN ENTEROCHROMAFFIN CELLS IN PLACE OF OR IN ADDITION TO 5-HYDROXYTRYPTAMINE

Appreciable azo coupling of enterochromaffin cells (EC) and of adrenal medulla (AM) is restricted to the alkaline range, fading out around pH 6, negative at 3-5. Low pH (3.0) azo coupling of rat and mouse mast cells (MC) with diazosafranin correlates with their known content of 5-hydroxytryptamine (5-HT) and with the pH 3-9 range of deep red azo coupling color reaction of p-nitrodiazobenzene with 5-hydroxytryptophan and 5-HT. The MC reaction is covalent azo bonding and not cationic dye salt bonding. The MC reaction is not influenced by prior oxidation by HIO4, FeCl3, K2Cr2O7 or I2/CH3OH exposures which prevent azo coupling of EC and of AM. The oxidation blockades of the azo coupling of EC and AM are reversed by Na2S2O5, N2S2O4 or Na2S2O3 reductions. These reductions after oxidation do not affect the MC diazosafranin reaction. In vitro the azo coupling of 5-HT is only slightly retarded but not weakened by HIO4 oxidation and then slightly enhanced by Na2S2O3, while noradrenaline gives red before oxidation, light yellow after, and after Na2S2O3, again deep red, the control α-naphthol giving deep red with p-nitrodiazobenzine at all three phases, before HIO4, between HIO4 and after Na2S2O3. Indole reactions for EC have been reported on glutaraldehyde (G), acetaldehyde and acrolein fixed but not formaldehyde (F) fixed guinea pig duodenum by Solcia and Sampietro (63), Geyer (27) (G), Barter and Pearse (5, 6) and Lillie and Greco-Henson (44). Repetition of the Lillie and Greco-Henson test of combining in the same preparation a blue indole reaction (postcoupled benzylidene) and a red azo coupling reaction with p-nitrodiazobenzene in both sequences disclosed numerous red EC and blue Paneth cells and eosinophil leukocytes, alike on F and G tissue. Direct observation during the second reaction and color photography of the same field after each of the two reactions showed that no red stained cell was replaced by a blue or purple one, and that no blue stained cell altered its color to purple or red. Moreover, azo positive cells were seen in the epithelium of the tips of the villi and in the sides of the gastric glands of the guinea pig pylorus and fundus. Indole positive cells did not occur in villus tip or pyloric gland epithelium and were restricted to fundus chief cells. It is concluded that the azo reactive substance in EC is a catechol and not 5-HT. The fluorescence studies have indicated the presence of 5-HT in EC, but the quantity present is insufficient for the acid azo coupling reaction shown by rat and mouse MC with diazosafranin.

The Staining of Brunner's Gland and Other Neutral Mucins by Carmine, Hematoxylin and Orcein in Alkaline Solutions

Brunners glands and other neutral mucins may be stained red, brownish red, and violet, respectively, by carmine, hematoxylin, and orcein from appropriate alkaline solutions. Carmine and hematoxylin in concentrations of 0.2-1% are dissolved in 60-70% alcohol containing 1% potassium carbonate; orcein is used in a 0.2% alcoholic solution of sodium hydroxide. Staining times are 15 to 30 minutes. The stained sections are rinsed in 95% or absolute alcohol prior to xylene and mounting. The staining of these mucins is blocked by mild bromine oxidation. By using alcian blue 0.1% in 3% acetic acid for 5 minutes prior to the above stains, mucins may be characterized in the same preparation as acid, neutral or mixed.

USE OF THE DIAZOSULFANILIC ACID, pH 1 AZURE A SEQUENCE ON ADRENAL MEDULLA AND THE EFFECTS OF PRIOR OXIDATION AND REDUCTION ON THE REACTION IN SEVERAL SPECIES

With the diazosulfanilic acid, pH 1 azure A sequence adrenal medulla exhibits areas of pale green cells with green nuclei alternating with other areas grading from green to dark blue. Adrenal cortex and capsular collagen color greenish yellow, nuclei and elastin green, smooth muscle yellowish green and erythrocytes greenish brown to greenish black. The reaction is obtained best after 5% glutaraldehyde, then 10% formol then glyoxal, quite well with formol-free sublimate and lead acetate fixations and fails after Zenker and Kose bichromate formol fixations. Staining is prevented by prior oxidation (quinonization) with 10% iodine/methanol (1-2 hr 3°C), 0.1 M FeCl3 (2 hr), 3-5% K2Cr2O7 (2-3 hr) and 1% H5IO6 (10 min) and after these oxidations is restored by 4 hr 5% dithionite (N2S2O4), metabisulfite (Na2S2O5) or thiosulfate (Na2S2O3 · 5H2O) reductions. The reaction has been demonstrated in man (Homo sapiens), monkey (Macaca mulatta), hog (Sus scrofa dom.), dog (Canis familiaris), cat (Felis cattus), guinea pig (Cavia cobaya), rat (Rattus norvegicus albinus), mouse (Mus musculus albinus), gerbil (Meriones unguiculatus) and opossum (Didelphis virginiana). In the hog most of the adrenal medulla gave the dark blue reaction, fairly large areas with some segregation into islets of dark blue occurred in man, monkey, cat, dog and opossum and islets are seen in mouse, smaller in rat and inconstantly in the guinea pig and gerbil. It is thought that the reaction is showing noradrenaline. The method is not specific for this substance; certain proteins as hemoglobin and keratohyalin as well as enterochromaffin cells react strongly.

Reduction and azo coupling of quinones

SummaryCutaneous melanin in formol fixed skin and adrenochrome in dichromate fixed monkey adrenal after adequate bisulfite or dithonite reduction were found to give definite azo coupling reactions. Weaker reactions were obtained on unreduced material, and these disappeared on ferric chloride oxidation. Both cutaneous melanin and adrenochrome appear to exist in a quinhydrone status. Prolongation of dichromate treatment weakens or abolishes azo coupling capacity of adrenochrome. The findings support the concept of quinonization and reduction to prevent and restore azo coupling of enterochromaffin cells and noradrenaline islets of the adrenal.The most effective diazos for melanin werep-nitrodiazobenzene, fast black K and the diazosulfanilic acid, pH 1 pyronin B procedure, for adrenochrome. Diazosafranin and 2-chloro-4-nitrodiazobenzene were also useful. Blue and violet coupling products from toluidine blue and methylene violet RR fail to yield sufficient contrast to be convincing.

Phosphomolybdic and phosphotungstic acid--Victoria Blue R stains two histochemically distinct collagens: dense dark blue and loose areolar pale green.

Victoria blue stains nuclei and elastic tissue but aqueous mounting media are required since stains are completely washed out by usual alcohol dehydration. Afterchroming with phosphomolybdic (PMo) or phosphotungstic (PW) acid converts the dye to an alcohol insoluble pigment. This is also insoluble in 1 N HCI and in acid alcohol. Premordanting with PMo or PW yields a staining pattern showing the uptake localization of these acids. A 2 to 3 mm 1% PMo or PW bath, followed by a 5 mm 1% Victoria blue R (VB) stain gives adequate coloration. Dense collagen of gastric submucosa and of derma colors dark blue, finely fibrillar areolar fibers and fat septa are light green (both stain red by Van Gieson), epithelia, keratin, gland cells and smooth and striated muscle stain light to moderate blue. Nuclei appear at most slightly deeper blue than cytoplasm, usually they are not identified. Cartilage is blue violet. Bromination, drastic acetylation, deamination and methylation inconsistently and only partially impair collagen staining. Methylation, as expected irreversibly blocks rat mast cell staining. When the PAS reaction is performed before the PMo-VB sequence, red staining is largely suppressed; when it follows the PMo-VB reaction it is well preserved, gastric mucin is deep red purple, basement membranes assume a deep purple tint. Cartilage may show mixed color or present blue pericellular areas in a red matrix. Interposition of the PMo-VB between the periodic acid and the Schiff reaction gives intermediate effects. Short acid dye counterstains (5-20 mm) may only impair areolar tissue stains. Orange G conferred some orange to yellow on muscle and erythrocytes, eosin B and Y had less effect, and Biebrich scarlet had more. A 24 hr 1% eosin gave: pink to red muscle, erythrocytes and epidermal keratin; light pink to pale green areolar tissue; moderate blue gastric glands and basal epidermis; and dark blue dense dermal and gastric submucosal collagen. The acid dyes probably react with the VB lake to form alcohol soluble neutral stains. Ranvier (1875) noted without details Krause’s staining with ammonium molybdate. Mallory (1891) introduced phosphomolybdic acid (PMo) hematoxylin as a connective tissue stain. Bethe ( 1896) used ammonium picrate and, better, ammonium molybdate to insolubiize and preserve Ehrlich’s supravital methylene blue for nerves and neurofibrillae. Mallory (1900) introduced the PMo aniline blue connective tissue stains and the phosphotungstic acid (PW) hematoxylin for fibrin, neuroglia and other objects. Serra and Queiroz-Lopez (1945) used molybdenum blue reduction for histochemical localization of tissue phosphates. Bunting (1951) simplified and modified this method which is quoted in detail by Lfflie (1954, 1976). Puchtler and Isler (1958) studied the effect of phosphomolybdic acid mordanting on rat intestine, staining with basic, amphoteric and acid dyes and comparing with standard connective tissue distribution and with the localization of molybdenum blue produced by stannous chloride reduction after phosphomolybdic acid mordanting. They concluded that the spec

THE pH RANGE OF THE DIAZOSAFRANIN REACTION OF RAT AND OTHER MAST CELLS

5-Hydroxytryptophan and 5-hydroxytryptamine (5-HT) give strong red azo coupling colors with fresh p-nitrodiazobenzene (fast red GG) at coupling pH levels of 3-9. Tyrosine, histidine, histamine, adrenaline, noradrenaline, dopa, dopamine and tryptophan give strong red colors at pH 7-9, weakening to orange and yellow at pH 5-6 and negative below. Tryptophan gives weak orange-yellow to about pH 4. Rat mast cells color deep red with diazosafranin at pH 3-8. Protein colors pink to red at pH 7-8, weaker at 5-6, and remains almost uncolored below that. Preoxidation with 10 min 1% H5IO6, 2 hr 0.1 M FeCl3, 3 hr 5% K2Cr2O7 or 1 hr 3°C 10% I2/CH3OH does not prevent the diazosafranin reaction of rat mast cells. Periodic acid does not inhibit in vitro azo coupling of 5-HT or 1-naphthol, that of noradrenaline is prevented, and can be restored by reduction with Na2S2O5 or Na2S2O3. Azo coupling of enterochromaffin and adrenal medulla is prevented by these oxidations and restored by Na2S2O4 reduction. Diazosafranin staining of mast cells is not extracted by 24 hr 0.24 N HCl/70% alcohol. Safranin and other cationic dye staining of rat mast cells resists aqueous 0.1 N HCl some hours, largely disappearing at 24 hr, and is removed by 5-15 min in 0.12 N HCl/7O% alcohol. At pH 1 0.1% toluidine blue colors rat mast cells deep violet; when superimposed after acid diazosafranin the red mast cells assume a deep purple, intermediate color. Since in extended use of the method pH 3 diazosafranin colors only bilirubin casts, hematoidin and dog, rat, mouse and gerbil mast cells, and not monkey, most human or lead and mercury fixed guinea pig mast cells, it is suggested that the method is showing 5-HT in rat mast cells. The occasional reaction of human mast cells may be due to pathologic presence of that substance in these cells.

Iron and Aluminum Lakes of Gallo Blue E As Nuclear and Metachromatic Mucin Stains

Gallo blue E, C. I. No. 51040, Mordant Violet 54, furnishes a blue black nuclear stain when applied to tissue sections in the form of its moderately stable iron lakes. This coloring combined well with such counterstains as orange G and eosin B. The Van Gieson stain tends to decolorize mucins, cartilage, and mast cells previously stained with this dye. Its aluminum lake solutions tend to gel in a few minutes to 24 hours depending on the solvent used and the amount of Al3+ present. Aluminum lake solutions give a moderately good blue to dark blue nuclear stain and a brilliant purplish red to dark purple stain to a variety of epithelial and connective tissue mucins. Acid dye counterstains are poorly tolerated. With either lake, nuclear staining is abolished by deoxyribonuclease digestion or relatively short mineral acid extraction of DNA.

Hematoxylin Substitutes: Fluorone Black and Methyl Fluorone Black (9-Phenyl- and 9-Methyl-2, 3, 7-Trihydroxy-6-Fluorone) as Metachrome Iron Alum Mordant Dyes

The phenyl and methyl trihydroxyfluorones, hitherto used histologically only in the rather difficult and unreliable Turchini technics for discriminating deoxyribonucleic from ribonucleic acid, find a new use as iron mordant metachrome dyes which act as nuclear stains. Nuclear staining is unaffected by acid extraction of nucleic acids, as with hematoxylin lakes. The two dyes, named by Liebermann and Lindenbaum 9-phenyl-2, 3, 7-trihydroxy-6-fluorone, have also acquired (illustrating with the phenyl homolog) longer chemical names of the form 2,6,7-trihydroxy-9-phenylisoxanthene-3-one (Eastman). Aldrich and Pfalz-Bauer adhere to the Liebermann-Lindenbaum nomenclature. The trivial name fluorone black is proposed for the phenyl homolog and methyl fluorone black for the methyl homolog. The iron lake of fluorone black appears to be a useful substitute for iron hematoxylin, methyl flurone black less useful. Neither dye has the diverse capability of hematoxylin.