Elastic Tissue Fibers Research Articles

Differential formation of a bioactive antiaggregating substance (BAS) by layers of the vein wall

Human umbilical veins were analyzed with and without endothelium in order to study the ability of endothelial cells and subendothelium to release a bioactive anti-aggregating substance (BAS: MW > 30kDa) independent of prostacyclin (PGI2). To evaluate the role of the subendothelium, the endothelial cells were removed by rubbing on filter paper for 1 min. We performed a histopathological study of the vessels using hematoxylin and eosine, and stained for elastic tissue fibers in order to confirm the presence of endothelium. The supernatant from incubated vascular rings was partially purified by Sephadex G-50 to rule out PGI2. The void volume fractions were collected and the anti-aggregating activity was tested on platelet aggregation induced by arachidonic acid, ADP, collagen and epinephrine. We observed that the activity was taking place with the use of the intact endothelium while there was no activity in the denuded vein. These observations could help to explain the well known antithrombotic properties of vascular endothelium.

Fluorescence and confocal laser scanning microscopy imaging of elastic fibers in hematoxylin-eosin stained sections.

We have studied the possibility of associating fluorescence microscopy and hematoxylin-eosin staining for the identification of elastic fibers in elastin-rich tissues. Elastic fibers and elastic laminae were consistently identified by the proposed procedure, which revealed itself to be easy and useful for the determination of such structures and their distribution. The fluorescence properties of stained elastic fibers are due to eosin staining as revealed by fluorescence analysis of the dye in solution, with no or only minor contribution by the elastin auto-fluorescence. The main advantage of this technique resides in the possibility of studying the distribution of elastic fibers in file material without further sectioning and staining. The use of the confocal laser scanning microscope greatly improved the resolution and selectivity of imaging elastic fibers in different tissues. The determination of the three-dimensional distribution and structure of elastic fiber and laminae using the confocal laser scanning microscope was evaluated and also produced excellent results. used to discriminate calcified bone and elastic fibers using the fluorescence microscope (Bradbeer et al. 1994). The selective fluorescence exhibited by these two main tissue components is based on the very faint eosin staining, while intensely eosinophilic substrates and those stained with the highly absorbing basic dyes show no fluorescence or just a very faint signal. Considering the possibility of using eosin fluorescence for the selective identification of some tissue components, we have extended this approach to the study of elastic fibers in hematoxylin-eosin (H&E) preparations. This paper then describes the results of observations with the fluorescence microscope of some H&E stained sections and evaluates the use of the confocal laser scanning microscope to image elastic fibers in the same preparations. Furthermore, we have also studied absorption and fluorescence spectra of eosin and phloxine, a closely related dye, in solution, to correlate them with the fluorescence detected in tissue sections.

Lysozyme Binds to Elastin and Protects Elastin from Elastase-Mediated Degradation

Lysozyme has been shown to be associated with damaged elastic fibers in many tissues and organs. To better characterize this interaction, binding of lysozyme to elastin was studied using solution-based binding assays. Under physiologic conditions, radio-labeled lysozyme bound specifically to elastin in a time- and concentration-dependent manner. Binding was reversible and was inhibited by unlabeled human and hen lysozyme but not by other proteins. Lysozyme had no elastolytic activity as assessed by a standard tritium-release assay, but, importantly, prevented the proteolytic degradation of elastin by human leukocyte elastase, pancreatic elastase, thermolysin, and Pseudomonas elastase. A striking feature of lysozyme's anti-elastase activity was that it did not function in the classical sense of inhibiting directly the enzymatic activity of the protease. Instead, by binding to elastin, lysozyme prevented the protease from interacting with the elastin substrate in ways that normally favor proteolysis. These results show that lysozyme binds to the elastin component of elastic fibers and that this interaction has important biological consequences for elastic fiber degradation. By preventing degradation of elastin, lysozyme can function as an important natural inhibitor that exerts a protective effect on elastic fibers at sites of tissue injury.

Elastic fibers in scar tissue.

The capacity of the skin to be stretched and to return to its resting position is correlated to the quantity and to the quality of the elastic fiber network. Although elastic fibers have been demonstrated in scars, the time course of their appearance in scars and their role in scar elasticity has not been elucidated. A study was therefore undertaken to evaluate the elastic fiber network in scars. The scars studied were from re-excision specimens following a biopsy performed for a benign or a malignant process. A total of 182 scars were evaluated in patients of different age groups. Miller's elastic tissue stain, considered to be superior to Verhoeff's van Giesen stain, was used. No elastic fibers were detected in any of 116 scars which were of less than 3 months' duration. In 66 scars present for over 3 months, a progressive increase in elastic fibers was present, first as focal and thin fibers, then as diffuse and thicker fibers. For scars of the same duration, a regional difference was noted in that scars from the back contained more and thicker elastic fibers than those from the cheek. When patients were stratified according to age, no appreciable difference was noted in the density of elastic fibers in both new and old scars between the different age groups. These results show that the synthesis of elastic tissue fibers in scars is a function of duration and site of the scar.

Elastoderma

Elastoderma is an exceedingly rare condition that is characterized clinically as an acquired localized laxity of skin resembling cutis laxa and histologically as an excessive accumulation of pleomorphic elastic structures within the dermis. We report the case of a 27-year-old white man with a 20year history of lax, extensible, wrinkled skin with areas of protrusion and ulceration localized to the anterior aspect and back of the neck. Histologic examination of specimens from the affected area revealed increased masses of intertwined thin, elastic fibers without calcification in the papillary and upper reticular dermis. Transmission and scanning electron microscopy demonstrated irregular deposition of elastic material at the periphery of elastic tissue fibers, with grapelike globular structures. To our knowledge, this is the second reported case of elastoderma.

Effect of low-grade conductive heating on vascular compliance during in vitro balloon angioplasty

Radiofrequency-powered, thermal balloon angioplasty is a new technique that enhances luminal dilatation with less dissection than conventional angioplasty. The purpose of this study was to assess the effect of radiofrequency heating of balloon fluid on the pressure-volume mechanics of in vitro balloon angioplasty and to determine the histologic basis for thermal-induced compliance changes. In vitro, radiofrequency-powered, thermal balloon angioplasty was performed on 46 paired iliac segments freshly harvested from 23 nonatherosclerotic pigs. Balloon inflations at 60°C were compared to room temperature inflations in paired arterial segments. Intraballoon pressure and volume were recorded during each inflation as volume infusion increased pressure over a 0 to 10 atm range. Pressure-volume compliance curves were plotted for all dilatations. Six segments were stained to assess the histologic abnormalities associated with thermal compliance changes. Radiofrequency heating acutely shifted the pressure-volume curves rightward in 20 of 23 iliac segments compared to nonheated controls. This increase in compliance persisted after heating and exceeded the maximum compliance shift caused by multiple nonheated inflations in a subset of arterial segments. Histologically, heated segments showed increased thinning and compression of the arterial wall, increased medial cell necrosis and altered elastic tissue fibers compared to nonheated specimens. In conclusion, radiofrequency heating of intraballoon fluid to 60°C acutely increases vascular compliance during in vitro balloon angioplasty of nonatherosclerotic iliac arteries. The increased compliance persists after heating and can be greater than the compliance shifts induced by multiple conventional dilatations. Arterial wall thinning and irreversible alteration of elastic tissue fibers probably account for thermal compliance changes.

Combination Hematoxylin-Eosin-Rose Bengal and Resorcin-Fuchsin Connective Tissue Stain for Glycolmethacrylate Embedded Sections

AbstractHematoxylin-eosin Y-rose bengal was combined with resorcin-fuchsin for the staining of glycolmethacrylate embedded tissue sections. In this procedure, resorcin-fuchsin staining is followed by Gill hematoxylin and eosin Y-rose bengal. Collagenous and elastic tissue fibers, as well as other tissue components, are clearly differentiated. (The J Histotechnol 16:141, 1993)

Low pressure radiofrequency balloon angioplasty: Evaluation in porcine peripheral arteries

Objectives. The purpose of this study was to evaluate the efficacy of radiofrequency-powered thermal balloon angioplasty in an in vivo porcine model.Background. Various modes of thermal energy used adjunctively during balloon angioplasty have demonstrated the potential to enhance the results of acute lumen dilation.Methods. In normal pigs, 75 peripheral arteries were dilated with a newly designed, radiofrequency-powered, thermal angioplasty balloon. All inflations were performed at 2-atm pressure for 85 s. Dilations were performed either with (hot) or without (cold) the application of heat. Lumen dimensions and vessel morphology were assessed with intravascular ultrasonography. At the end of each study, dilated arterial segments were harvested for histologic examination.Results. Single cold balloon inflations resulted in a 12.7% increase in arterial cross-sectional area whereas single hot inflations resulted in a 22.9% increase (p < 0.03). Similarly, when multiple cold inflations were compared with multiple hot inflations, two, three and four sequential hot inflations resulted in a significantly greater increase in cross-sectional area than an equivalent number of cold inflations (p < 0.03).Histologic examination demonstrated a temperaturedependent effect on the depth of medial necrosis and extent of arterial wall thinning (p < 0.001) as well as evidence for uniform alteration of elastic tissue fibers at temperatures of ≥60 °C (p < 0.03).Conclusions. Low pressure radiofrequency thermal balloon angioplasty results in a greater increase in cross-sectional area in porcine peripheral arteries than does nonheated conventional balloon angioplasty. The pathologic basis for this enhanced dilation may be a temperature-dependent effect on medial necrosis, thinning of the arterial wall or alteration of vascular elastic fibers, alone or in combination.

Tumor necrosis factor-alpha down-regulates human elastin gene expression. Evidence for the role of AP-1 in the suppression of promoter activity.

Cytokine modulation of elastin gene expression was examined by assay of elastin mRNA abundance and by transient transfections of cultured human skin fibroblasts and rat aortic smooth muscle cells with elastin promoter/reporter gene (chloramphenicol acetyltransferase, CAT) constructs. Incubation of cells with human recombinant tumor necrosis factor-alpha (TNF-alpha) markedly suppressed the elastin mRNA levels in a time- and dose-dependent manner by up to 91%. TNF-alpha also suppressed the expression of the elastin promoter/CAT construct by up to 70% in transiently transfected cells, indicating regulation at the transcriptional level. This suppression was temporally preceded by rapid and transient up-regulation of c-jun and c-fos genes. The down-regulatory effect of TNF-alpha on elastin promoter activity was abolished by co-transfections with a synthetic double-stranded AP-1 oligomer. Furthermore, co-transfection of the elastin promoter construct with c-jun and c-fos expression plasmids resulted in a marked decrease in the promoter activity. Elucidation of the cis-regulatory elements in the elastin promoter by 5' deletion construct analysis implicated a region -290 to -198 containing one AP-1 binding site. The functional role of this AP-1 site was further tested by gel retardation assays which indicated formation of a DNA-protein complex specific for TNF-alpha treated cells. This complex could be partially dissociated by a competing oligomer containing the consensus AP-1 binding site. These observations suggest that the inhibitory effects of TNF-alpha on elastin gene expression involve the transcription factor AP-1. Interferon-gamma also suppressed the elastin gene expression at the mRNA level by approximately 52%, but it had no effect on the elastin promoter activity, suggesting post-transcriptional mechanisms. These results indicate that mediators released from inflammatory cells can modulate elastin gene expression, and such modulation may play a role in diseases characterized by altered accumulation of elastic fibers in tissues.

Tissue Vitronectin in Normal Adult Human Dermis Is Non-Covalently Bound to Elastic Tissue

Vitronectin is a multifunctional human plasma glycoprotein that is also found in constant association with elastic tissue fibers in normal adults. We have investigated the nature of the association of vitronectin with elastic tissue, and compared it to that of other elastic fiber-associated proteins, namely fibrillin and amyloid P component. Samples of normal human dermis were incubated with a variety of extraction agents, including high molar salt solution, non-ionic detergent (Nonidet P-40), the reducing agents dithiothreitol or 2-mercaptoethanol, and the chaotropic agents sodium dodecyl sulfate or guanidine hydrochloride. Vitronectin purified from serum typically migrates as two bands of 75 and 65 kD. By contrast, immunoblotting studies of residual dermal material after extraction with the various agents revealed only lower molecular weight (58, 50, 42, 35, and 27 kD) anti-vitronectin reactive bands. Although these bands may represent degradation products of vitronectin generated as a result of the extraction procedure, we cannot exclude the possibility that tissue vitronectin is distinct from plasma vitronectin. Anti-vitronectin reactive polypeptides co-migrating with the 58-, 50-, and 42-kD bands were solubilized following extraction with sodium dodecyl sulfate or guanidine hydrochloride, but not with the other extraction agents. Immunofluorescence studies using residual dermal material after extraction with guanidine hydrochloride demonstrated a marked reduction in elastic fiber staining intensity with anti-vitronectin and anti-amyloid P component, but not with anti-fibrillin. Thus the majority, if not all of dermal vitronectin, is, like amyloid P component, non-covalently associated with, and not an integral constituent of, elastic fibers.

Eosinophil degranulation. An immunologic determinant in the pathogenesis of the Mazzotti reaction in human onchocerciasis.

Onchocerciasis patients treated with diethylcarbamazine often undergo a severe inflammatory response, the Mazzotti reaction. To assess the eosinophil's role in the pathogenesis of the Mazzotti reaction, we obtained serial blood, plasma, and skin biopsy specimens from 21 heavily infected patients and 3 endemic controls, both before and during therapy with diethylcarbamazine. Samples were analyzed for blood eosinophils, plasma levels of eosinophil granule major basic protein (MBP) and eosinophil-derived neurotoxin, eosinophil infiltration and eosinophil and mast cell degranulation in the skin. After the first dose of diethylcarbamazine, blood eosinophils fell from a pre-treatment level of 888 +/- 111 to 203 +/- 42 cells/mm3 at 8 h. This decrease was followed by a marked eosinophilia developing over the remaining 7 days of treatment and 14 days of follow-up. Plasma eosinophil-derived neurotoxin levels increased from 56 +/- 4 ng/ml pretreatment to a peak of 82 +/- 9 ng/ml at 8 h and returned to pretreatment levels by 48 h. Beginning at 12 h, plasma MBP levels increased from 730 +/- 74 ng/ml pretreatment to a peak of 1140 +/- 74 ng/ml after 5 days. Pretreatment skin biopsies stained for MBP by immunofluorescence showed a bright fibrillar pattern in the dermis consistent with chronic eosinophil degranulation; the MBP was localized on elastic tissue fibers. After treatment, skin biopsy specimens showed both the pretreatment fibrillar MBP staining pattern as well as focal eosinophil degranulation. Deposition of MBP around microfilariae in the papillary dermis was visible as early as 1.5 h. The lowest blood eosinophil levels and peak plasma eosinophil-derived neurotoxin levels coincided with the infiltration and degranulation of eosinophils in the skin. Mast cell degranulation in the skin was maximal by the first posttreatment biopsy (1.5 h) coincident with the beginning of eosinophil degranulation. Although the pathogenesis of the Mazzotti reaction is clearly complex, our results indicate that eosinophil degranulation is characteristic of the response and that it occurs with a time course suggestive of a role for the eosinophil in determining the clinical and pathologic manifestations of the reaction.

Distribution of putative elastic fibers in rabbit temporomandibular joint tissues.

This study describes the general distribution of putative elastic fibers in the connective tissues that comprise the articular disk and some of the adnexal tissues of the rabbit temporomandibular joint. Joints were removed en bloc and processed for light-microscopic study. The fibroelastic tissues of the bilaminar zone of the articular disk and the various attachments of the disk to the mandibular condyle contained numerous elastic fibers. Since these morphologic data indicate the presence of many elastic fibers, we suggest that the rabbit temporomandibular joint may serve as a model system to study the functional consequences of selectively altering the quality or quantity of elastic fibers in these tissues.

Morphometric analysis of the growth of the normal fetal guinea pig lung.

The structural development of the fetal guinea pig lung is described and quantified morphometrically in this report. At 35 days gestation the lung is in the pseudoglandular phase of growth, by 40 days it is in the canalicular phase, and at 50 days the saccular growth phase has begun. At term (67 days), the fetal guinea pig lung appears mature. From the beginning of the canalicular to the end of the saccular phases, the correlation coefficient between lung volume and gestational age is +.98, between internal surface area and gestational age is +.94 and between total number of saccules and gestational age is +.97. Internal surface area (ISA) correlates closely with lung volume (r = +.99) and the correlation coefficient between total number of saccules and lung volume is +.98. At term, lung volume is 4.22 ml. ISA is 0.5 M2, and total number of saccules is 253 million. Parenchymal growth is achieved by increases in both number and size of airspaces in the canalicular phase, primarily by increases in number during the early saccular phase and largely by increases in airspace size near term. The total length of parenchymal elastic tissue increases from 223 M at 45 days gestation to 5,253 M at term. Elastic tissue fibers first appear in the parenchyma of the fetal guinea pig lung during the canalicular phase, when the rate of saccule formation is high. The quantitative increase in elastic tissue correlates closely with the increase in the total number of saccules from day 45 to day 60 of gestation (r = +.99). The rate of elastic tissue growth increases sharply in the late saccular phase, coinciding with the period of greatest saccular expansion. These data suggest an interdependent relationship between saccular growth, i.e., proliferation and expansion, and the development of lung parenchymal elastic tissue.

Ultrastructural and elemental analysis of calcification of advanced swine aortic atherosclerosis

During progression and in the early phase on a regression regimen, calcificition of the necrotic protion of the atheroma of swine abdominal aorta occurred primarily in degenerated cells or in membranous, vesicular cellular degradation products which varied in size, shape, and the amount of mineral deposit. Calcium appeared to be deposited in amorphous granular or needle-like crystalline forms. Energy dispersive X-ray and line profile analysis showed that the major elements in the heavily calcified portions of the plaques were calcium and phosphorus. There was a direct relationship between the distribution and concentration of these elements indicating that the mineral deposit was a calcium phosphate. Select area electron diffraction analysis of grossly calcified portions of the plaque gave a diffraction pattern identical to that of calcium hydroxyapatite. Calcification was not observed to occur on elastic tissue or collagen fibers.

“ANATOMIC SEQUELAE OF BALLOON ANGIOPLASTY OF PULMONARY ARTERIES IN CHILDREN”

Three children, in whom seven pulmonary arteries (PA)had been balloon dilated, were evaluated at autopsy. Ages at dilation were 2½, 2½, and 8 years. The dilating balloon was three to four times the diameter of the undilated PA. Dilating pressure was7-9 atmospheres. In five PA, angiographically measured predilation diameter averaged 2.6mm(2-2.9mm) and post dilation diameter averaged 6mm (5.5-6.6mm). In two PA,balloon dilation had occurred zero and three days prior. Each PA revealed a long,linear,medial tear. Microscopy revealed transmedial rupture and hemorrhage into the media and adventitia of both. The other five PA were evaluated 4-14 months post-dilation. Each PA had a long,linear,healed, medial tear. Microscopy revealed prior transmedial rupture in all 5 PA.Healing occurred by reconstitution of media to approximately normal thickness in 4 of 5. This neo media was composed of disrupted and noncircumferential elastic tissue fibres interspersed with collagen. In one PA, 14 months post dilation, the media at the rupture site was only lmm thick. Increased collagen and elastic fibre deposition was present in the adventitia in all five and also in the intima in two. These healing processes did not compromise PA diameter. Transmedial rupture was the major mechanism of diameter increase in all 7 PA. In six the adventitia maintained PA integrity. One PA ruptured during balloon dilation.

A rapid digestive technique to expose networks of vascular elastic fibers for SEM observation.

The NaOH sonication digestion technique permits rapid isolation and exposure of intact networks of elastic fibers in vascular tissue for 3-dimensional observation with the SEM. The configuration of the network of elastic fibers within the vascular wall of large elastic arteries (aorta) is generally agreed to be a flexible framework through which smooth muscle cells and collagenous fibers are interwoven. However, the configuration of elastic fiber networks in muscular arteries, medium sized veins and smaller vessels remains unknown. When the lengthy standard biochemical elastin purification techniques were applied to vessels containing lesser amounts of elastic tissue and finer elastic fibers, the vessels were completely digested. In contrast, the digestion and sonication technique isolated and exposed intact networks of delicate elastic fibers in blood vessels which do not contain large amounts of elastic tissue. Unfixed vessels were cut into short segments, placed in 0.5 N NaOH and sonicated for 20-40 min. The specimens were rinsed in deionized distilled H2O, then autoclaved for 30 min. The tissue was rinsed a second time, fixed and processed routinely for SEM. Elastic stains and enzymatic digestion with chromatographically purified elastase and collagenase confirmed that the digestion and sonication technique produced clean, isolated networks of elastic fibers. Knowledge of the configuration of the networks of elastic fibers in different vessels enhances understanding of distensibility characteristics of individual vessels and serves as a baseline for studying alterations in the elastic framework which occur during aging and disease processes such as atherosclerosis, arterial hypertension and aneurysms.

Immunohistochemical demonstration of amyloid P component in skin of normal subjects and patients with cutaneous amyloidosis.

The presence of amyloid P component (AP) in dermal deposits of cutaneous amyloidosis was demonstrated by a direct immunofluorescence technique using an antibody to serum amyloid P component (SAP). AP was also shown, for the first time, to be a constituent of normal human skin. It was present at the periphery of dermal elastic tissue fibres, in basement membranes of dermal blood vessels and surrounding eccrine sweat glands but was absent from the dermo-epidermal basement membrane. The staining pattern in cutaneous amyloidosis was morphologically distinctive and readily distinguishable from staining of thickened vascular basement membranes in porphyria. Immunofluorescence with anti-SAP is simple and specific and may become the procedure of choice in the differential diagnosis of amyloidosis.

The histomorphology of the elastic tissue system in the skin of the human hand

This study compares the effectiveness of the Verhoeff and Puchtler-Sweat resorcin fuschin methods of staining the elastic tissue of palmar and dorsal skin specimens of the human hand. The resorcin stain demonstrates the "oxytalan" fibres of the elastic tissue system in the dermal papillary layer of the palmar skin. Oxytalan fibres are not generally demonstrated by the Verhoeff method and are not demonstrated in the dorsal skin specimens. These studies substantiate the existence of a system of elastic tissue fibres in the skin of the hand. The inter-relationship of the elastic and collagenous fibres appears to complement each of their functions; namely, in anchoring the dermo-epidermal junction, absorbing the stresses of stretch and compressive forces and return the tissues to their original state of tension, and providing protection to the dermal appendages in addition to lending a gradient mobility transmitted through the dermis to the subcutaneous layer to give suppleness and mobility to the skin.

Biochemistry of the Elastic Fibers in Normal Connective Tissues and its Alterations in Diseases

The elastic fibers present in various connective tissues of the body are responsible for physiologic elasticity of the organs. These fibers consist of 2 distinct components, elastin and the elastic fiber microfibrils. Controlled synthesis and balanced interaction of these 2 components are essential for normal fibrillogenesis. The intracellular biosynthesis of elastin by connective tissue cells, such as smooth muscle cells, involves assembly of the polypeptide chains on the membrane-bound ribosomes, hydroxylation of some prolyl residues to hydroxyproline, and secretion of the polypeptides packaged in Golgi vacuoles. In the extracellular space the elastin molecules assemble into fiber structures which are stabilized by the synthesis of complex covalent cross-links, desmosines. Recently, aberrations in the structure or metabolism of elastin have been detected in a variety of heritable and acquired diseases affecting skin and other connective tissues. These conditions include pseudoxanthoma elasticum, cutis laxa, and elastosis perforans serpiginosa, as well as arteriosclerosis and other degenerative changes of the vascular connective tissues.

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.