Keratin in the Egg Shells of Amphistomes; Histochemical Differentiation from Quinonetanned Protein in Other Trematoda

Various combinations of the oxidation method for demonstrating keratin in shell material of amphistomes were tried. Acidified permanganate worked more efficiently than performic and peracetic acids, and Alcian blue and aldehyde fuchsin excelled other basic dyes for subsequent staining. For the permanganate-Alcian blue reaction, sections of material fixed in Susa or Bouin were oxidized in 0.3% permanganate in 0.3% H₂SO₄ for 5 min., decolourized in 1% oxalic acid, stained in 3% Alcian blue in 2 N H₂SO₄ and counterstained with eosin. The shell globules stained a deep blue. For permanganate aldehyde fuchsin staining, the sections were stained in aldehyde fuchsin for 1 hr, after oxidation with permanganate. The shell globules then stained a deep magenta. The catechol and fast red reactions were negative in amphistomes and the specimens lack the characteristic amber colour due to quinone tanning.     

The intragranular location of carboxyl groups in neuromelanin and lipofuscin in human brain and in meningeal melanosomes in mouse brain

The intragranular location of carboxyl groups was tinctorially determined in human substantia nigra neuromelanin granules, human inferior olive lipofuscin granules, and mouse meningeal melanosomes. Soluble and insoluble lipid was stained with beta naphthol Sudans in unoxidized and oxidized frozen and paraffin sections containing neuromelanin or lipofuscin. Nile blue demonstrated carboxyls in unoxidized neuromelanin, lipofuscin, and melanin, and in oxidized neuromelanin and lipofuscin. Carbodiimide demonstrated carboxyls in unoxidized and oxidized lipofuscin and oxidized neuromelanin. In all instances, staining for carboxyls was inhibited by prior mild methylation, and proof of their presence was obtained by a pre-staining, stepwise, alternating, and repetitive mild demethylation, mild methylation sequence. Structurally, carboxyls were demonstrated in the neuromelanin granule's soluble lipid-free lipofuscin component, in the meningeal melanosome's melanin component, and virtually throughout the lipofuscin granule. The following structural and chemical basis was proposed for the different resistance of Nile blue staining of melanosomes and of neuromelanin and lipofuscin to acetone extraction. Nile blue forms an insoluble complex with melanosomal dopa-melanin's quinonoid, diphenolic, and undissociated carboxyl units. Such complex formation does not occur in neuromelanin's carboxyl-free dopamine-melanin component, however. Instead, Nile blue ionogenicly bonds with dissociated carboxyls belonging to the neuromelanin granule's lipofuscin component.     

Quantitative Determination of Murine Dermal Elastic Fibers by Color Image Analysis: Comparison of Three Staining Methods

We compared three different staining methods to determine if the dermal elastic fiber content of the HRS/Skh-1 hairless mouse could be accurately measured by color image analysis. Comparisons were made among Klig-man's modification of Luna's mast cell stain for elastin, Unna's orcein stain with or without potassium permanganate preoxidation, and Gomori's aldehyde fuchsin stain with potassium permanganate preoxidation. The color image analysis system could be used to identify and quantify murine dermal elastin fibers in sections stained by all three methods. Gomori's aldehyde fuchsin stain with preoxidation demonstrated twice the content of dermal elastic fibers demonstrated by either Kligman's modification of Luna's mast cell stain or Unna's orcein stain with or without preoxidation. Gomori's aldehyde fuchsin method with preoxidation should be considered the stain of choice for evaluating murine dermal elastic fiber content.     

Studies on the molecular arrangement of RNA in tissues with a selective topo-optical reaction of RNA

Summary Selective, demonstration of RNA in tissues was achieved by treating tissue sections with potassium permanganate followed by bisulfite and toluidine blue at pH. 1.0 (PBT reaction). It is suggested that this reaction is due to aldehyde groups which are formed by the oxidative cleavage of the pyrimidine rings of RNA which can be selectively demonstrated using bisulfite-toluidine blue as the aldehyde reagent.The specificity of the reaction was tested after RNAase treatment, after acid hydrolysis, and on pure RNA droplets. The aldehyde nature of the reacting groups was checked, after permanganate oxidation, by Schiff's leucofuchsin reagent, and by aldehyde blocking reactions.Two types of intracellular molecular arrangement of RNA molecules could be distinguished by polarization optics after application of the PBT reaction: 1) The strong birefringence, dichroism and metachromatic staining of membrane-bound RNA in ergastoplasm of pancreas, liver and plasma cells indicate a linear (planar) molecular order of RNA molecules on the surface of the membranes, and 2) the isotropic, basophilic staining of RNA not organized in membrane structures (Nissl substance, nucleoli) suggest a random distribution of their dye binding sites.     

Studies on the molecular arrangement of RNA in tissues with a selective topo-optical reaction of RNA.

Selective demonstration of RNA in tissues was achieved by treating tissue sections with potassium permanganate followed by bisulfite and toluidine blue at pH 1.0 (PBT reaction). It is suggested that this reaction is due to aldehyde groups which are formed by the oxidative cleavage of the pyrimidine rings of RNA which can be selectively demonstrated using bisulfite-toluidine blue as the aldehyde reagent. The specificity of the reaction was tested after RNAase treatment, after acid hydrolysis, and on pure RNA droplets. The aldehyde nature of the reacting groups was checked, after permanganate oxidation, by Schiff's leucofuchsin reagent, and by aldehyde blocking reactions. Two types of intracellular molecular arrangement of RNA molecules could be distinguished by polarization optics after application of the PBT reaction: 1) The strong birefringence, dichroism and metachromatic staining of membrane-bound RNA in ergastoplasm of pancreas, liver and plasma cells indicate a linear (planar) molecular order of RNA molecules on the surface of the membranes, and 2) the isotropic, basophilic staining of RNA not organized in membrane structures (Nissl substance, nucleoli) suggest a random distribution of their dye binding sites.     

Early secretory activity in the hypothalamic nuclei and neurohypophysis in the rat; determined by selective staining

Visualization of stainable material in the neural lobe of the rat provided the most reliable index of the age at which secretory activity can first be recognized, though preceded by both hypothalamic synthesis and axonal transportation. A problem of interpretation was encountered in the neural lobes of fetal and infant animals, due to different staining responses obtained during this age period, to the two methods of staining employed; chrome alum hematoxylin-phloxin and aldehyde fuchsin after oxidation by either acidified potassium permanganate or performic acid. With aldehyde fuchsin the material of the neural lobe is stainable selectively from the eighteenth day of fetal life to adulthood. With hematoxylin phloxine the first staining response also occurred in the posterior lobe but much later, at the end of the first postnatal week. The staining situation in the pars neuralis has its counterpart in the differentiating hypothalamic nuclei; complicated by the differentiation of the supraoptic nuclei some days in advance of the paraventricular nuclei. After aldehyde fuchsin staining, evidences of neurosecretory activity were present in the perikarya of the supraoptic nuclei at birth, but mature neurons were rarely seen in the paraventricularis until at least 24 hours later. Nuclei of fetal hypothalami were not studied, but the demonstration of stainable material in the fetal neural lobes constitutes circumstantial evidence of functional competence of some neurons of either one or both types of nucleus, most likely the supraoptic.     

An investigation of aldehyde fuchsin staining of unoxidized insulin

Aldehyde fuchsin is a standard stain for the secretion granules of pancreatic B cells. The participation of either insulin or proinsulin in aldehyde fuchsin staining is in dispute. There is some evidence that permanganate oxidized insulin is stained by aldehyde fuchsin. Aldehyde fuchsin staining of unoxidized insulin has not been investigated adequately despite excellent staining results with tissue sections. Unoxidized insulin and proinsulin suspended by electrophoresis in polyacrylamide gels were fixed with Bouin's fluid and placed in aldehyde fuchsin for one hour. Because the unoxidized proteins were not stained by aldehyde fuchsin, it was concluded that neither insulin or proinsulin are responsible for the intense aldehyde fuchsin staining of unoxidized pancreatic B cell granules in tissue sections. A series of controlled experiments was undertaken to test the effects of fixatives, oxidation and destaining procedures on aldehyde fuchsin staining of insulin, proinsulin and other proteins immobilized in polyacrylamide gels. It was demonstrated that only oxidized proteins were stained by aldehyde fuchsin and that cystine content of the proteins had no apparent relation to aldehyde fuchsin staining. It was concluded that neither insulin nor proinsulin is likely to be responsible for the intense aldehyde fuchsin staining of unoxidized pancreatic B cell granules in tissue sections.     

The demonstration of two acid groups in juxtaglomerular granules with basic triphenyl methane dyes,aldehyde-fuchsin and schiff's reagent

Summary Oxidation and bromination of mouse kidney JG cell-granules result in the production of cysteic acid from cystine; cysteic acid is capable of taking up rapidly and selectively certain basic triphenyl methane dyes including aldehyde fuchsin at lower pH levels.After treatment with periodic acid, bromine and hydrochloric acid, the JG granules or the nuclear chromatin also take up the basic triphenyl methane dyes (including aldehyde fuchsin) which contain amino groups, probable as a result of the production of aldehyde groups. Basic triphenyl methane lacking amino groups does not react with aldehydes.Some substance present in JG granules could be stained by aldehyde fuchsin after prior oxidation; HCl methyl violet 2B was taken up both with or without prior oxidation. Only strong methylation completely abolished these affinities which were restored after demethylation. These reactions are attributed to cystine.The staining of JG granules with dilute aldehyde fuchsin and dilute methyl violet 2B is not affected by oxidation, bromination, aldehyde blocking and hydrolysis; these reactions are abolished by mild methylation, but restored by subsequent saponification. These staining properties are due to the presence of carboxylic acid in JG granules.The positive PAS reaction of JG granules is due to the presence of 1.2-glycol in the same granules.     

Aldehyde fuchsin staining, direct or after oxidation: problems and remedies, with special reference to human pancreatic B cells, pituitaries, and elastic fibers.

Successful production of aldehyde fuchsin (AF) having the unique properties described by Gomori depends on each of many critical variables. AF made from basic fuchsins which contain mainly rosanilin (C.I. 42510) do not stain properly-fixed pancreatic B cells, pituitary basophils, or elastic fibers in unoxidized sections. AF made from basic fuchsins containing mainly pararosanilin (C.I. 42500) stains these entities strongly. Substances stained by AF without oxidation fall into two classes: 1) nonacidic peptides and proteins, most of which contain half-cystines, and 2) polyanions, particularly when sulfated. Group 2 substances stain rapidly, Group 1 substances stain slowly. Many modifications of aldehyde fuchsin have been described. Modified aldehyde fuchsins (MAFs) differ in the kind of aldehyde and in the amount of aldehyde and hydrochloric acid used in their formulation; they differ also in the temperature and duration of the ripening necessary before they can be used. If microsections are first oxidized by acid permanganate or other oxidant, MAF staining of pancreatic B cells, pituitary basophils and other substances containing cystines is speeded and intensified. Most modified methods prescribe oxidation, but the author's does not. The chemical basis, final result and potential side-reactions of oxidation methods (OXMAF) differ from those of direct methods (DIMAF) such as the author's. DIMAF staining is slower but inherently simpler and less destructive. The time required for optimal staining with DIMAF depends on the potency of the stain, which in turn depends on how the stain was made and its age. Detection of DIMAF--reactive peptides and proteins may be hampered by the strong staining of polyanions. This can be remedied if the polyanions are first stained with Alcian blue (AB) or other durable basic dye of contrasting color resistant to acid ethanol. Experiences with the AB-DIMAF staining of pancreatic B cells, pituitaries and elastic fibers in formalin-fixed human tissues are detailed. Proper control of the variables which affect MAF will insure useful and reliable results either directly or after oxidation. Authors and editors are urged to be more careful hereafter to distinguish the results of DIMAF from those of OXMAF methods. Published reports should always specify the parameters that affect the properties of MAF. In OXMAF methods the steps intervening between oxidation and staining should be spelled out. Such care should help dispel the confusion and uncertainty which cloud the use and reputation of aldehyde fuchsin at present. This unique dye deserves wider and wiser use.     

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.     

The role of paraldehyde in the rapid preparation of aldehyde fuchsin

Preparation of aldehyde fuchsin normally requires ripening for 3 to 5 days. By using a 5-fold excess of paraldehyde a fully potent aldehyde fuchsin can be prepared in 24 hr at room temperature. Aldehyde fuchsin prepared by both normal and accelerated ripening afforded comparable results, including selective staining of unoxidized pancreatic B cells. Dried aldehyde fuchsin prepared form pararosaniline and reconstituted in acid alcohol has spectrophotometric properties different form the ripened strain. Reconstituted aldehyde fuchsin stains unoxidized B cells adequately only if staining time is extended. Excess paraldehyde added to reconstituted aldehyde fuchsin retards decomposition but does not produce a normal stain by spectrophotometric standards. Warming of aldehyde fuchsin solutions to accelerate ripening has been shown to produce deleterious effects and should be avoided.     

A Staining Sequence for A, B and D Cells of Pancreatic Islets

Recent investigations strongly suggest the elaboration of a third pancreatic hormone by the D cell and the existence of cells which show the staining properties of both B and D cells. Demonstration of these and all other islet cells in a single section is possible by the following staining sequence: (1) of D cells by silver or toluidine blue, (2) of B cells by pseudoisocyanin, and (3) empirical staining of all islet cells together by aldehyde fuchsin, ponceau de xylidine, acid fuchsin and light green. Difficulties in embedding compact pancreatic tissue can be overcome by dehydrating to 80% ethanol, followed by tetrahydrofurane as the intermediate fluid to paraffin infiltration.     

Basic Fuchsin in Acid Alcohol: A Simplified Alternative to Schiff Reagent

The use of Schiff reagent to demonstrate polysaccharides (after prior periodic oxidation) and nucleic acids (after prior acid hydrolysis) is unnecessary since the same results are obtained by substituting a 20 min staining in a 0.5% w/v solution of basic fuchsin in acid alcohol (ethanol-water-concentrated HC1, 80:20:1) followed by a rinse in alcohol. The shade of the basic fuchsin staining is a little yellower than that achieved with Schiff reagent but the selectivity, light fastness, response to different fixatives, and to prior histo-chemical blocking of the tissue section were much the same for the two methods. The need for prior oxidation or hydrolysis and the inhibitory effect of aldehyde blocking techniques indicate that basic fuchsin, like Schiff reagent, reacts with aldehyde groups. Infrared studies indicate that for cellulose the reaction product is an azomethine.     

Effects of Streptomyces hyaluronidase digestion on the histochemical reactions of proteoglycans in cartilage compared with its effects on certain non-cartilaginous tissues

Summary To test the value ofStreptomyces hyaluronidase in carbohydrate histochemistry, the effects of digestion with the enzyme on the staining of cartilage and non-cartilaginous tissues by Alcian Blue (AB) pH 1.0, AB pH 2.5, high iron diamine, low iron diamine, aldehyde fuchsin, dialysed iron-ferrocyanide and AB pH 2.5-periodic acid-Schiff were studied by light microscopy. The results obtained lead to the conclusion that theStreptomyces enzyme releases not only hyaluronic acid but also chondroitin sulphates and keratan sulphates in cartilage. Since hyaluronic acid is known to be linked to chondroitin sulphate proteoglycans, the enzyme is of limited value in localizing hyaluronic acid in cartilage. However, it is useful in localizing hyaluronic acid in most non-cartilaginous tissues.     

Silver Impregnation of Alzheimer's Neurofibrillary Changes Counterstained for Basophilic Material and Lipofuscin Pigment

A method is described in which selective silver staining of Alzheimer's neurofibrillary changes is combined with staining of cell nuclei, Nissl material, and lipofuscin granules. Formalin fixed, paraffin embedded sections of human autopsy tissue are silver stained according to a method proposed by Gallyas. Lipofuscin is stained by crotonaldehyde fuchsin following performic acid oxidation. Nissl substance is visualized by either Darrow red or gallocyanin-chrome alum staining. Architectonic units showing the specific pathology and the neuronal types prone to develop the neurofibrillary changes can be recognized using this technique.     

Silver impregnation of Alzheimer's neurofibrillary changes counterstained for basophilic material and lipofuscin pigment

A method is described in which selective silver staining of Alzheimer's neurofibrillary changes is combined with staining of cell nuclei, Nissl material, and lipofuscin granules. Formalin fixed, paraffin embedded sections of human autopsy tissue are silver stained according to a method proposed by Gallyas. Lipofuscin is stained by crotonaldehyde fuchsin following performic acid oxidation. Nissl substance is visualized by either Darrow red or gallocyanin-chrome alum staining. Architectonic units showing the specific pathology and the neuronal types prone to develop the neurofibrillary changes can be recognized using this technique.     

Histochemical Comparison of the Casella, Bauer, and Periodic Acid Oxidation-Schiff Leucofuchsin Technics

Chromic acid, potassium permanganate and periodic acid apparently produce aldehyde from the same general group of substances. Chromic acid and potassium permanganate also destroy the aldehyde which they have produced, as well as that previously produced by periodic acid oxidation and by the Feulgen hydrochloric acid hydrolysis. Sulfite blockade, by occupying a considerable proportion of the aldehyde groups produced by periodic acid oxidation, creates a Schiff reaction similar to that produced by primary or secondary chromic acid or permanganate oxidation.

It is suggested that the periodic acid Schiff positive substances which fail to give distinct Bauer and Casella reactions, are those with relatively few reactive 1,2 glycol, 1,2 OH,NH₂, or 1,2 OH,NHR groupings per molecule.     

Histochemical Comparison of the Casella,Bauer, and Periodic Acid Oxidation-Schiff Leucofuchsin Technics

Chromic acid, potassium permanganate and periodic acid apparently produce aldehyde from the same general group of substances. Chromic acid and potassium permanganate also destroy the aldehyde which they have produced, as well as that previously produced by periodic acid oxidation and by the Feulgen hydrochloric acid hydrolysis. Sulfite blockade, by occupying a considerable proportion of the aldehyde groups produced by periodic acid oxidation, creates a Schiff reaction similar to that produced by primary or secondary chromic acid or permanganate oxidation.

It is suggested that the periodic acid Schiff positive substances which fail to give distinct Bauer and Casella reactions, are those with relatively few reactive 1,2 glycol, 1,2 OH,NH₂, or 1,2 OH,NHR groupings per molecule.     

Sulfite suppresses the mutagenic property of coffee

The mutagenicity of instant and freshly brewed coffee on Salmonella typhimurium TA100 and TA98 without S9 mix was inactivated by sodium sulfite. Sulfite ion at a dose of 200 ppm almost completely inactivated the mutagenicity of coffee made in the ordinary way (5-15 mg dry weight/ml). Sodium bisulfite and potassium metabisulfite had similar effects. On the contrary, L-ascorbic acid enhanced the mutagenicity of coffee. Sodium sulfite also inactivated the phage-inducing activity of coffee in inductest III. Sodium sulfite completely suppressed the mutagenicities of 1,2-dicarbonyls, namely diacetyl and glyoxal. Diacetyl is present in coffee, beer, butter and other foods and drinks. Because sodium sulfite, sodium bisulfite and potassium metabisulfite are widely used as food additives, they should be useful in reducing the levels of mutagens in foods.