Faye Sweat

ON THE BINDING OF CONGO RED BY AMYLOID

Congo red in alkaline 80% ethyl alcohol solution saturated with NaCl stains amyloid selectively. The distribution of the dye is very similar to that observed in serial sections stained with Highmans Congo red method. In sections stained with Congo red in aqueous or alcoholic solutions without differentiation all tissue structures are intensely colored, but only amyloid is dichroic. The rapid removal of dye by alkaline solutions from structures other than amyloid suggests ionic or salt-type bonds between Congo red and these structures. In contrast, the binding of Congo red by amyloid from alkaline alcoholic solutions and the increase in intensity of staining upon addition of NaCl indicate a non-ionic type linkage between amyloid and dye. The effects of deamination, acetylation and various oxidation procedures on the binding of Congo red indicate interaction of the dye with hydroxyl groups of amyloid. According to data on the direct dyeing of cellulose in textile dyeing, Congo red is adsorbed by hydrogen bonding between hydroxyl groups of the polysaccharide chains and the amino groups of the dye. It seems probable that a similar mechanism is involved in the binding of Congo red by amyloid. It is suggested that the selective staining of amyloid with Congo red is due to steric configurations of amyloid or one of its components which favor non-ionic binding of the eye.

Investigation of staining, polarization and fluorescence-microscopic properties of myoendothelial cells

Electron microscopists have described in vascular endothelial cells filaments which resemble myofibrils. These fibrils cannot be demonstrated with conventional staining techniques. On the basis of previous investigations of relations between dye structure and affinity for muscle fibres, a staining method for demonstration of myoendothelial cells by direct, polarization and fluorescence microscopy has been developed. Carnoy‐fixed paraffin sections were treated consecutively with Kernechtrot, tannic and phosphomolybdic acid and counter‐stained with Levanol fast cyanine 5RN. This procedure stained myoendothelial cells and muscle fibres deep blue, connective tissues yellow and nuclei pink. For polarization and fluorescence‐microscopic studies thiazine red R was substituted for Levanol fast cyanine 5RN and Kernechtrot was replaced by Mayers acid hæmalum. The light‐microscopic characteristics of fibrils in endothelial cells and in smooth muscle cells were identical.

Histochemical specifity of staining methods for connective tissue fibers: Resorcin-fuchsin and van Gieson's picro-fuchsin

Conclusions and summaryDepending on the pretreatment of tissue sections, resorcin-fuchsin stained collagen, reticulum fibers and/or basement membranes and ring fibers intensely. It must therefore be concluded that resorcin-fuchsin is not specific for the protein elastin, that is elastic fibers in the chemical sense of the term.Studies of van Gieson-type stains showed relation between dye structure and affinity for connective tissue fibers. These observations are in good agreement with data from textile and leather dyeing that the behaviour of sulfonated dyes is largely determined by the configuration of the dye molecule and the presence of additional reactive groups.Polarization microscopic studies of stained sections — based on data derived from textile chemistry — demonstrated the possibility to obtain information concerning the submicroscopic structure of tissue components. Several methods, though still in the experimental stage, have been found valuable for the study of pathological lesions of connective tissue. Because electron microscopy is unsuitable for routine histopathology, it is highly desirable to develop convenient methods for the study of submicroscopic structures in general hospital pathology.

A review of early concepts of amyloid in context with contemporary chemical literature from 1839 to 1859.

Early concepts of amyloid are reviewed in context with chemical literature from 1839-1859. Cellulose was first described in 1839. Treatment with sulfuric acid converted cellulose into a substance which was colored blue by iodine. This cellulose derivative was called amyloid. Virchow applied the chemical tests for starch and cellulose to human tissues. The substance in waxy degeneration reacted like immature cellulose. Virchow did not coin the term amyloid for this substance but suggested it as a compromise. Virchow restricted the term amyloid to the carbohydrate moiety and explicitly excluded the protein fraction which he had described earlier. Friedreich and Kekulé (1859) studied an evidently impure sample of amyloid and concluded that it did not contain polysaccharides. Since then the protein moiety has been studied extensively, but the carbohydrate fraction has been largely neglected. It appears possible that investigations of the carbohydrates at sites of amyloid formation would aid the understanding of the pathogenesis of amyloidosis2.

ON THE BINDING OF DIRECT COTTON DYES BY AMYLOID

Application of 30 direct cotton dyes in aqueous salt solution to amyloid in human tissues yielded different staining patterns. Only dyes with good wool reserve without reactive metal atoms, i.e., dyes which stain cellulose but not proteins or polyamides, colored amyloid selectively without differentiation. Direct dyes for union fabrics, i.e., dyes which color cellulose and protein fibers, stained all tissue structures. The effects of deamination and acetylation on the binding of direct dyes with good wool reserve indicate interaction of these dyes with hydroxyl groups of amyloid. The increase in intensity of staining upon addition of NaCl to the dye solution suggests a nonionic type linkage. According to chemical data on the direct dyeing of cellulose, direct dyes are adsorbed by hydrogen bonding between hydroxyl groups of the polysaccharide chains and suitable groups in the dye. It seems probable that a similar mechanism is involved in the binding of these dyes by amyloid. The selective staining of amyloid with direct dyes with good wool reserve, which do not color proteins, suggests that the polysaccharide moiety of amyloid may play a major role in the binding of direct dyes.

ON THE MECHANISM OF RESORCIN-FUCHSIN STAINING

Acetylation, sulfation and phosphorylation induce binding of resorcin-fuchsin by glycogen, basement membranes, reticulum fibers, collagen, and other tissue structures containing polysaccharides as demonstrated by the periodic acid Schiff reaction. These structures do not stain with resorcin-fuchsin in control section without pretreatment. It is concluded that the binding of resorcin-fuchsin is due to the introduction of ester groups. Extraction procedures, designed to remove dyes held by salt-like or ionic linkage, indicate binding of resorcin-fuchsin by non-ionic bonds. According to data on the dyeing of the polysaccharide ester cellulose acetate in textile dyeing, cellulose acetate dyes are adsorbed by hydrogen-bonding between the carbonyl oxygen of the ester group and phenolic hydroxyl groups of the dye. It seems possible that in resorcin-fuchsin a phenolic hydroxyl group of the resorcinol moiety of the dye is free to react. The intense staining of agar, esterified glycogen, collagen and reticulum fibers with resorcin-fuchsin implies that binding of this dye does not convey any information concerning the protein moiety of tissue structures. Thus, resorcin-fuchsin can not be considered specific for elastic fibers in the chemical sense of the term.

A Modified One-Step Trichrome Stain for Demonstration of Fine Connective Tissue Fibers

Gomoris one-step trichrome procedure was modified to improve coloration of fine connective tissue fibers. Paraffin sections from tissues fixed in alcohol, acetone, Zenkerformol, 10% formalin, Kaiserlings or Carnoys fluid were mordanted 1 hr at 56 C in Bouins solution, stained 1 min in a trichrome solution (chromotrope 2R-phosphomolybdic acidaniline blue WS) adjusted to pH 1.3 with HCl, rinsed in 1% aqueous acetic acid, dehydrated and covered. Collagen, reticulum fibers, basement membranes, ring fibers around splenic sinuses, intercalated discs in cardiac muscle and cartilage were colored blue. Nuclei, cytoplasm, fibrin, muscle fibers and elastic fibers were stained red. Pretreatment of sections with Bouins solution enhanced the affinity of tissues for chromotrope 2R and was found essential for satisfactory coloration of material fixed in alcohol, acetone, formalin or Carnoys fluid. Because this method does not require differentiation, it gave uniform results even in the hands of inexperienced labora...

A Selective Stain for Renal Basement Membranes

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

On the mechanism of sequence iron-hematein stains.

SummaryAs a prerequisite for the histochemical study of sequence iron-“hematoxylin” stains the iron alum-acidified hematein procedure was developed which does not require differentiation.Histochemical blocking and extraction procedures demonstrated that carboxyl and hydroxyl groups are essential for the binding of cationic iron.The iron alum-Prussian blue reaction colored collagen, reticulum fibers and basement membranes more intensely than muscle fibers. Treatment of tissue sections mordanted in iron alum with the acidified hematein solvent resulted in practically complete removal of iron from all tissue structures. It must therefore be concluded that the selective staining of muscle fibers, terminal bars and related structures with sequence iron-hematein stains is not due to high affinity of iron for these tissue components.Observations by R. and M. Heidenhain on sequence hematoxylin-potassium dichromate and hematoxylin-alum stains and data from modern textile chemistry indicate that the staining patterns obtained with metal-hematein sequence stains are determined by the affinity of the hematein moiety for certain tissue structures.

A cresyl fast violet stain for bacteria and fungi in tissue.

The cresyl fast violet staining method was modified to eliminate differentiation. Paraffin sections from tissues fixed in Zenker-formol were stained in a 1% aqueous solution of cresyl fast violet (Chroma), adjusted to pH 3.7 with acetic acid, washed in running tap water, dehydrated and covered. Because basophilia increases with time of fixation or storage in formalin or Kaiserlings fluid, dilution of the dye solution to 0.5-0.1% is recommended for such material. Bacteria, nuclei, Nissl substance, and lipofuscin were colored dark blue; fungi, blue to purple; and cytoplasm and muscle fibers, light blue. Collagen and reticulum fibers were only faintly stained. Thus, microorganisms were easily visible against the lightly colored background. In formalin-fixed material, bile pigment was colored olive green. Because this method does not require differentiation, it gave uniform results even in the hands of different users. Little or no fading was observed in sections stored for more than 2 yr.