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.     

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.     

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.     

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.     

Only aldehyde fuchsin made from pararosanilin stains pancreatic B cell granules and elastic fibers in unoxidized microsections: problems caused by mislabelling of certain basic fuchsins

The most distinctive property of aldehyde fuchsin is its staining of certain nonionic proteins and peptides in unoxidized cells and tissues. These substances include granules of pancreatic islet B cells, elastic fibers and hepatitis B surface antigen. Aldehyde fuchsin made from two different basic fuchsins, each certified by the Biological Stain Commission and labelled C.I. (Colour Index) No. 42500 (pararosanilin), did not stain pancreatic B cells at all. Stain Commission's records and retesting showed that each of the "faulty" basic fuchsins was not pararosanilin, but rosanilin, whose Colour Index number is 42510. These basic fuchsins were labelled with the wrong Colour Index number when packaged. Additional basic fuchsins were coded by V.M.E. and tested by R.W.M. for their capacity to make satisfactory aldehyde fuchsins. Only certain of these aldehyde fuchsins stained unoxidized pancreatic islet B cells. The same aldehyde fuchsins stained elastic fibers strongly. Each basic fuchsin whose aldehyde fuchsin was judged satisfactory proved to be pararosanilin. Aldehyde fuchsin solutions made from other basic fuchsins stained elastic fibers only weakly and did not stain pancreatic B cells at all in unoxidized sections. Each basic fuchsin whose aldehyde fuchsin was unsatisfactory proved to be rosanilin. It appears that only aldehyde fuchsin made from pararosanilin stains unoxidized pancreatic B cell granules dependably. We found that basic fuchsins from additional lots of Commission-certified pararosanilin and rosanilin were also labelled with incorrect Colour Index numbers when packaged. Steps were taken to prevent recurrences of such mislabelling which has made it difficult until now to correlate differences in the properties of pararosanilin and rosanilin. A table is provided of all basic fuchsins that have been certified by the Biological Stain Commission since 1963 when they began the practice of subdesignating basic fuchsins according to whether they are pararosanilins or nonpararosanilins. The consumer can readily determine from the certification number on the label the correct subdesignation of any Commission-certified basic fuchsin listed here. Until now, mislabelling of some lots of pararosanilin as rosanilin and vice-versa has confused and frustrated the users of basic fuchsins in other applications such as the carbol fuchsin staining of tubercle bacilli and certain cytochemical tests, e.g. esterase and acid phosphatase, that utilize hexazotized pararosanilin as a coupling reagent. Consumers experiencing trouble with any Commission-certified dye should look to the Biological Stain Commission for help. This is an important reason for purchasing, whenever possible, only Biological Stain Commission certified dyes.     

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.     

Simplified staining in situ of neurosecretory cells of the supraoesophageal ganglion of the female Culex pipiens molestus (Forskal) (Diptera: Culicidae)

The author describes the morphology and distribution of the neurosecretory cells in the supraoesophageal ganglion of the adult female Culex pipiens molestus, using paraldehyde fuchsin and paraldehyde thionine-paraldehyde fuchsin as vital staining techniques. The brain of Culex pipiens molestus has three regions (the proto-, deuto- and tritocerebrum) in which principally two types of neurosecretory cells (A and B) can be detected. Both A (dark) and B (light) cells are to be found in the protocerebrum, where they are termed medial neurosecretory cells, as they are distributed in the pars intercerebralis and only a few occur more laterally. A small group of type A and B neurosecretory cells is to be found in the posterior part of the brain (the tritocerebrum). These cells display characteristics differences in their tinctorial affinity.     

Effects of different fuchsin analogs on the Feulgen reaction

The Feulgen reaction is used for cytophotometric quantitation of nuclear DNA. Schiff's reagents used in the Feulgen reaction usually are prepared from basic fuchsin, a variable mixture of four triaminotriphenylmethane analogs. The effect of the several fuchsin analogs on the quality of Schiff's staining of hydrolyzed DNA is not known. In this investigation Schiff's reagents prepared from relatively pure fuchsin analogs were used to determine whether different fuchsin analogs affect the absorbance of the Schiff's reagent-DNA complexes formed in solution. It has been determined that the complex formed by pararosaniline-Schiff's reagent and hydrolyzed DNA exhibits lower absorption than do corresponding complexes formed by Schiff's reagents prepared from magenta II or from new fuchsin.     

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 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 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 Demonstration of Beta Cells in Pancreatic Islets and Their Tumors

The stain is applied routinely to tissues fixed in 10% buffered formalin (pH near 7.0) or in Bouin's fluid. Bring paraffin section to water as usual and mordant 72 hr in 5% CrCl₃ dissolved in 5% acetic acid. Wash in water and in 70% alcohol and stain 6 hr. Formula of staining solution: new fuchsin, 1% in 70% alcohol, 100 ml; HCl, conc., 2 ml and paraldehyde, 2 ml, mixed together and added to the dye solution; let stand 24 hr before use. After staining, wash in running tap water 5-10 min, rinse in distilled water and counterstain if desired. Dehydration in alcohol, clearing and covering completes the process. When the paraldehyde is obtained from a freshly opened bottle, standardized staining times can be used and thus eliminate the necessity of differentiating individual slides. The granules of beta cells stained deep blue to purple and were demonstrated in the pancreatic islet of man, dog, mouse, frog, guinea pig and rabbit.     

Granular cells in the connective tissue of Helix pomatia L. (Gastropoda,Pulmonata)

Summary Granular cells (cells crowded with colourless granules staining with paraldehyde fuchsin according to Gomori-Gabe and not containing calcium) are independent cells in the connective tissue of Helix pomatia. Histochemical data suggest that the granules are rich in sulfhydryl-containing proteins, but lack biogenic monoamines. Electron microscopic investigations confirm the supposed secretory activity of the granular cells. Secretory proteins are presumed to be synthetized in the endoplasmic reticulum and condensed in the Golgi apparatus giving rise to the granules. Extrusion occurs by exocytosis.Electrophoresis of homogenates, prepared from tissues containing numerous granular cells, results in the separation and identification of a secretory protein from the granular cells. An electrophoretically homologous protein is recognized in the hemolymph, but in very small quantities.Our findings and the work of others suggest the involvement of granular cells in neuroendocrine events.The author is indebted to Prof. Dr. D. Kuhlmann for suggesting the problem and for his valuable criticism during the investigation. I would like to thank Mr. J.N. Howell, who helped with the English.Part of this work has been supported by the Stiftung Volkswagenwerk and by the Deutsche Forschungsgemeinschaft     

Resorcin Fuchsin Staining of Neurosecretory Cells

The resorcin fuchsin staining solution was prepared by dissolving 1 gm of the dry dye (Chroma) in 98 ml of 70% ethanol acidified by 2 ml of concentrated HCI. When applied to paraffin sections of vertebrate hypothalamus fixed in a modified Bouin's fluid (0.5% trichloroacetic acid replacing 5% acetic), the solution stained neurosecretory cells in a manner comparable to staining by Gomori's aldehyde fuchsin. The resorcin fuchsin solution requires no ripening and is said to keep for months. It showed no deterioration in the 20 day period of testing. Optional fixatives are: unmodified Bouin's, Heidenhain's SUSA, and alcoholic trichloroacetic acid.     

A histochemical investigation of the mechanism of Aldehyde Fuchsin staining of pancreatic B-cell granules

Summary The reaction mechanism by which Aldehyde Fuchsin selectively stains pancreatic B-cell granules is unknown. The participation of either insulin or proinsulin in the reaction is debatable; the stain may be bound by other components of the B-cell granule or its membrane. Sections of pancreas were stained with a variety of basic stains and specific histochemical reagents with and without appropriate blocking agents. No evidence for strong tissue anions associated with the B-cell granule could be found. Aldehyde Fuchsin staining was not abolished by lowering the pH below the point at which all known tissue anions should be protonated. There was no evidence that the Aldehyde Fuchsin staining solution itself generates reactive groups in the tissue. The results of this investigation support a non-ionic, possibly covalent mechanism for Aldehyde Fuchsin staining of pancreatic B-cell granules.     

A Historical Perspective on the Identification of Cell Types in Pancreatic Islets of Langerhans by Staining and Histochemical Techniques

Before the middle of the previous century, cell types of the pancreatic islets of Langerhans were identified primarily on the basis of their color reactions with histological dyes. At that time, the chemical basis for the staining properties of islet cells in relation to the identity, chemistry and structure of their hormones was not fully understood. Nevertheless, the definitive islet cell types that secrete glucagon, insulin, and somatostatin (A, B, and D cells, respectively) could reliably be differentiated from each other with staining protocols that involved variations of one or more tinctorial techniques, such as the Mallory-Heidenhain azan trichrome, chromium hematoxylin and phloxine, aldehyde fuchsin, and silver impregnation methods, which were popularly used until supplanted by immunohistochemical techniques. Before antibody-based staining methods, the most bona fide histochemical techniques for the identification of islet B cells were based on the detection of sulfhydryl and disulfide groups of insulin. The application of the classical islet tinctorial staining methods for pathophysiological studies and physiological experiments was fundamental to our understanding of islet architecture and the physiological roles of A and B cells in glucose regulation and diabetes.     

Demonstration of Elastic Tissue and Iron or Elastic Tissue and Calcium Simultaneously

For the concomitant demonstration of iron and elastic tissue Perls' test solution was used, followed by Verhoeff's stain or Gomori's aldehyde fuchsin. When Perls' and Verhoeff's stain were used in sequence, the iron deposits were greenish blue and the elastic lamellae were black. When Perls' test solution was combined with aldehyde fuchsin the iron deposits were blue and elastic tissue purple. Calcium salts and elastic tissue were demonstrated concomitantly by using von Kossa's method followed by Gomori's aldehyde fuchsin. With such combined staining, the calcium salts appeared brownish black and elastic tissue purple. With these procedures, it was possible to see the exact relationship of calcium and iron deposits to the elastic tissue.     

Demonstration of Elastic Tissue and Iron or Elastic Tissue and Calcium Simultaneously

For the concomitant demonstration of iron and elastic tissue Perls' test solution was used, followed by Verhoeff's stain or Gomori's aldehyde fuchsin. When Perls' and Verhoeff's stain were used in sequence, the iron deposits were greenish blue and the elastic lamellae were black. When Perls' test solution was combined with aldehyde fuchsin the iron deposits were blue and elastic tissue purple. Calcium salts and elastic tissue were demonstrated concomitantly by using von Kossa's method followed by Gomori's aldehyde fuchsin. With such combined staining, the calcium salts appeared brownish black and elastic tissue purple. With these procedures, it was possible to see the exact relationship of calcium and iron deposits to the elastic tissue.     

Specificity and Sensitivity of Insulin Staining by Aldehyde Fuchsin, Pseudoisocyanin and Toluidine Blue

Aldehyde fuchsin, pseudoisocyanin and toluidine blue, histochemical dyes reported to be specific for insulin-containing granules of the pancreatic beta cell, were applied to insulin fixed in polyacrylamide gel by disc electrophoresis. Two major and four minor bands were resolved as demonstrated by staining with amidoschwarz; only the two major bands, were stained by aldehyde fuchsin. The addition of serum did not affect this reaction. Serum or insulin components gave no metachromatic reactions to the other stains. Under the conditions applied, aldehyde fuchsin is the only one of these dyes specific for insulin in this, system, but this stain is not sufficiently sensitive to detect normal serum levels of the hormone.     

Morphological changes of pancreatic B cells in ventromedial hypothalamic (VMH)-lesioned obese rats

Morphological changes of pancreatic B cells were investigated in ventromedial hypothalamic (VMH)-lesioned obese rats. An increase in body weight, a decrease in body length, and a marked increase in fat-pad were observed in VMH-lesioned obese rats. The volume density of pancreatic islets and that of B cells in VMH-lesioned obese rats were more increased than those in sham-operated control rats. The B cells of VMH-lesioned obese rats often showed a slight reaction for aldehyde fuchsin or anti-pig insulin serum. By the electron microscopy, the degranulated B cells were found to contain well developed Golgi apparatus and rough endoplasmic reticulum. These observations indicate increased activities of synthesis and release of insulin. We concluded that VMH-lesioned obesity was caused by accelerated lipogenesis with hypersecretion of insulin.