Similarities between stratum corneum basic protein and histidine-rich protein II from newborn rat epidermis

The stratum corneum basic protein and histidine-rich protein II were each isolated from newborn rat epidermis and compared by biochemical and immunologic methods. The proteins were indistinguishable by immunodiffusion using antiserum elicited to either protein. The migration of the proteins on SDS-polyacrylamide gel electrophoresis was identical giving a molecular weight of 49 000. These proteins, which have similar but unusual amino acid compositions, give very similar tryptic peptide maps. Both proteins aggregate with keratin filaments to form macrofibrils. These results suggest that histidine-rich protein II and stratum corneum basic protein are the same protein. We suggest that this protein be called histidine-rich basic protein.     

Identification of a rapidly labelled 350K histidine-rich protein in neonatal mouse epidermis

The major histidine-rich protein (HRP) found in the stratum corneum of neonatal mouse epidermis (band 2 protein, molecular weight 27,000) is a relatively late product of epidermal differentiation and incorporates labelled amino acids in vivo only after a 6-9 h lag period. A number of putative precursor HRPs in the 70-300 K molecular weight range were initially identified using short pulse labeling times and our previously described methods for isolation of epidermis and extraction of proteins. However, when steps were taken to minimise proteolysis during preparation, a single species of approximately 350 K molecular weight was the most strongly labelled protein following a 1 h in vivo pulse of [3H]-histidine. This protein was stable in sodium dodecyl sulphate dithiothreitol at 100 degrees C and in 4 M urea, suggesting a single covalently linked polypeptide. The kinetics of labelling and the localisation of the 350 K HRP in the lower granular layers suggest that it is a precursor of the stratum corneum HRP. The processing of the 350 K HRP to the stratum corneum species appears to involve a complex series of specific cleavage steps which give rise to a number of HRPs of intermediate molecular weight.     

Identification of a Rapidly Labelled 350K Histidine-Rich Protein in Neonatal Mouse Epidermis

Abstract. The major histidine-rich protein (HRP) found in the stratum corneum of neonatal mouse epidermis (band 2 protein, molecular weight 27,000) is a relatively late product of epidermal differentiation and incorporates labelled amino acids in vivo only after a 6–9 h lag period. A number of putative precursor HRPs in the 70–300 K molecular weight range were initially identified using short pulse labelling times and our previously described methods for isolation of epidermis and extraction of proteins. However, when steps were taken to minimise proteolysis during preparation, a single species of approximately 350 K molecular weight was the most strongly labelled protein following a 1 h in vivo pulse of [³H]-histidine. This protein was stable in sodium dodecyl sulphate dithiothreitol at 100°C and in 4 M urea, suggesting a single covalently linked polypeptide. The kinetics of labelling and the localisation of the 350 K HRP in the lower granular layers suggest that it is a precursor of the stratum corneum HRP. The processing of the 350 K HRP to the stratum corneum species appears to involve a complex series of specific cleavage steps which give rise to a number of HRPs of intermediate molecular weight.     

The characterization of human epidermal filaggrin. A histidine-rich, keratin filament-aggregating protein

Filaggrin is a histidine-rich, cationic protein that aggregates with keratin filaments in vitro and may function as the keratin matrix protein in the terminally differentiated cells of the epidermis. This protein has been previously isolated from rodent epidermis. In this investigation, a similar protein from human skin was identified, isolated and characterized by biochemical and immunologic techniques. Indirect immunofluorescence of human skin using antiserum to rat filaggrin gave positive immunofluorescence of keratohyalin granules and the stratum corneum. This indicated the presence of a human filaggrin in the epidermis in a localization similar to that of the rodent. The protein was isolated from human epidermis and purified by ion-exchange chromatography and preparative gel electrophoresis. The purified protein crossreacts with antibody to rat filaggrin and migrates as a doublet of molecular weight (Mr) approximately 35 000 on SDS-polyacrylamide gels. It is relatively rich in polar amino acids such as histidine, arginine, serine and glycine, but is poor in nonpolar amino acids. Unlike rodent filaggrin, the human protein contains ornithine. This protein aggregates with human keratin filaments, forming compact macrofibrils in a manner analogous to that of rodent filaggrin. Thus, a human epidermal protein has been isolated which has many of the characteristics of rodent filaggrin and may function as the human keratin matrix protein.     

Evidence of a precursor form of stratum corneum basic protein in rat epidermis.

The fully differentiated anucleate cells of the stratum corneum of newborn rat epidermis contain a cationic protein called stratum corneum basic protein (SCBP). This protein has a molecular weight (49 000) and an amino acid composition similar to a protein extracted from the less differentiated cell layers of the epidermis. Pulse--chase experiments with radiolabeled histidine were undertaken to test the possiblity that SCBP is derived from a preexisting protein. A protein of 52 000 daltons is rapidly but transiently labeled in extracts of the less differentiated cell layers. As the amount of label in the 52 000-dalton protein decreases, an increase in radiolabel is observed in extracts of the fully differentiated cells. This label is found in SCBP, a protein of lower molecular weight (49 000) than that initially labeled. These proteins are immunologically related and both are resistant to cyanogen bromide cleavage. They differ in apparent molecular weight on sodium dodecyl sulfate--polyacrylamide gels and in their net charge. The results are consistent with the conversion of a precursor protein into SCBP.     

Biosynthetic pathways of filaggrin and loricrin--two major proteins expressed by terminally differentiated epidermal keratinocytes

We have used immunoelectron microscopy to map the biosynthetic pathways of loricrin and filaggrin in epidermal keratinocytes at successive stages of differentiation in newborn mouse skin. The filaggrin epitope is first detected in large, irregularly shaped, keratohyalin granules (F-granules) in the stratum granulosum, and then distributed throughout the cytoplasms of the innermost layers of stratum corneum cells. We conclude that the poly-protein filaggrin precursor is first accumulated in F-granules, from which it is subsequently released and processed into filaggrin, and becomes associated with the densely packed bundles of keratin filaments inside stratum corneum cells. Its diminished visibility in the outer layers correlates with the known degradation of filaggrin to free amino acids. Loricrin is first detected in small round keratohyalin granules (L-granules), and subsequently at the periphery of cells throughout the stratum corneum. Labeling of purified keratinocyte envelopes establishes that this loricrin epitope is exposed only at their inner (cytoplasmic) surface. Thus loricrin is initially accumulated in L-granules, to be released at a specifically programmed stage of keratinocyte maturation, and incorporated into the covalently cross-linked lining of the cell envelope. Since loricrin is rich in cysteine, L-granules account for the sulfur-rich keratohyalin granules described earlier. Proposals are made to rationalize why, subsequent to synthesis, filaggrin precursor and loricrin should be segregated both from each other and from the rest of the cytoplasm.     

Putative histidin-rich proteins in the epidermis of lizards

In the stratum granulosum of mammalian epidermis, histidin-rich proteins (filaggrins) determine keratin clumping and matrix formation into terminal keratinocytes of the stratum corneum. The nature of matrix, interkeratin proteins in the epidermis of nonmammalian vertebrates, and in particular in that of reptilian, mammalian progenitors are unknown. The present biochemical study is the first to address this problem. During a specific period of the renewal phase of the epidermis of lizards and during epidermal regeneration, keratohyalin-like granules are formed, at which time they take up tritiated histidine. The latter also accumulate in cells of the alpha-keratin layer (soft keratin). This pattern of histidine incorporation resembles that seen in keratohyalin granules of the stratum granulosum of mammalian epidermis. After injection of tritiated histidine, we have analysed the distribution of the radioactivity by histoautoradiography and electrophoretic gel autoradiography of epidermal proteins. Extraction and electrophoretic separation of interfilamentous matrix proteins from regenerating epidermis 3-48 hours post-injection reveals the appearance of protein bands at 65-70, 55-58, 40-43, 30-33, 25-27, and 20-22 kDa. Much weaker bands were seen at 100, 140-160, and 200 kDa. A weak band at 20-22 kDa or no bands at all are seen in the normal epidermis in resting phase and in the dermis. In regenerating epidermis at 22 and 48 hours post-injection, little variation in bands is detectable, but low molecular weight bands tend to increase slightly, suggesting metabolic turnover. Using anti-filaggrin antibodies against rat, human, or mouse filaggrins, some cross-reactivity was seen with more reactive bands at 40-42 and 33 kDa, but it was reduced or absent at 140, 95-100, 65-70, 50-55, and 25 kDa. This suggests that different intermediate degradative proteins of lizard epidermis may share some epitopes with mammalian filaggrins and are different from keratins with molecular weight ranging from 40 to 65-68 kDa. The immunocytochemical observation confirms that a weak filaggrin-like immunoreactivity characterizes differentiating alpha-keratogenic layers in normal and regenerating tail. A weak filaggrin labeling is discernable in small keratohyalin-like granules but is absent from the larger granules and from mature keratinocytes. The present results indicate, for the first time, that histidine-rich proteins are involved in the process of alpha-keratinization in reptilian epidermis. The cationic, interkeratin matrix proteins implicated may be fundamentally similar in both theropsid-derived and sauropsid amniotes.     

Protein synthesis during fetal development of mouse epidermis : I. The appearance of “histidine-rich protein”

The synthesis of specific proteins at different stages in the development of mouse epidermis was investigated. The epidermis from mice of an age range from 16 days of fetal life to 7 weeks postpartum was prepared by maceration in acetic acid, and the total proteins were separated by SDS-polyacrylamide gel electrophoresis. The appearance of particular proteins at different developmental stages was correlated with the morphology of the epidermis at the same stage. Several qualitative and quantitative changes in the protein patterns were observed during development, the significance of which is discussed. A protein of molecular weight approximately 27,000 was the major component of a group of closely related proteins which appeared simultaneously with the production of mature keratohyalin granula in 18-day fetal mouse epidermis. Isolation and characterization of this protein showed it to be a “histidine-rich protein.” Its concentration in the epidermis decreased as the number of keratohyalin granula diminished with increasing age. The protein could be localized exclusively in the keratohyalin granula by a fluorescence method involving staining with dansyl chloride. This method was also used to detect “histidine-rich protein” on polyacrylamide gels.     

Epidermolytic toxin binds to components in the epidermis of a resistant species

Ferritin-labeled epidermolytic toxin selectively bound to resin-impregnated sections of toxin-resistant rat skin. Binding was confined to the keratohyalin granules of the stratum granulosum and to the cytoplasm of cells in the stratum corneum. Single granules, and equivalent regions in composite granules, failed to bind the conjugate. Preferential binding of the ferritin-toxin probe created anastomosed arrays in the large granules. The scoring density within corneal cells varied with the position of the cell in the stratum corneum. It reached a maximum in the fourth cell from the granular-corneal junction, then decreased to a negligible value in the outermost corneal layers. Three groups of toxin-binding polypeptides were identified in epidermal extracts. These include profilaggrin, a group of three histone H1 polypeptides and histone H3. Filaggrin did not react with toxin on Western blots. The findings demonstrate that sensitivity to epidermolytic toxin is not solely dependent on the presence of target material. It is suggested that species-specific differences in the morphology of keratohyalin granules and in metabolism of profilaggrin may be important factors in intoxication.     

Endogenous lectin from terminally differentiated epidermal cells

We isolated a concanavalin A (Con-A)-binding glycoprotein from human stratum corneum by nonionic detergent extraction, lectin affinity chromatography, and preparative gel electrophoresis. This glycoprotein migrates as a single band at 40 kilodaltons at sodium-dodecyl-sulfate gel electrophoresis with or without the presence of 2-mercaptoethanol. It was shown to have a heterogeneous distribution between pH 5.6 and 7.6 by isoelectric focusing. The glycoprotein is histidine rich (10.4%) but is distinct from other histidine-rich proteins (epidermal filaggrin and the histidine-rich glycoprotein from serum). It does not bind to lectins specific for L-fucose or alpha-D-galactose. We prepared a monospecific polyclonal antibody to the 40-kilodalton glycoprotein; at the ultrastructural level, it cytoimmunolocalizes exclusively to the membranes of the stratum corneum. A unique feature of the glycoprotein is that it is an endogenous lectin: it hemagglutinates trypsinized and gluteraldehyde-fixed rabbit erythrocytes. The inhibition of its hemagglutination was found to be greatest with amino sugars, down to a saccharide concentration of 10(-5) mM. Such a high affinity of binding at the cell surface suggests that this glycoprotein is a major carbohydrate-binding, cross-linking molecule that holds adjacent corneocytes together in the stratum corneum. We hypothesize that this lectin plays a role in the adhesion and desquamation of the stratum corneum.     

Two polypeptide chain constituents of the major protein of the cornified layer of newborn rat epidermis.

The insoluble component of stratum corneum of rat epidermis yields two major bands after extraction with 8 M urea-mercaptoethanol-dithiothreitol. The ratio of these two bands is about 1:1 in terms of protein stain intensity and S-[14C]carboxymethyl label. Both polypeptides were purified to homogeneity by DE-52-cellulose, sodium dodecyl sulfate hydroxylapatite C column chromatography, and preparative DodSO4-polyacrylamide gel electrophoresis. The heavier polypeptide contains 30% alpha helix and the lighter contains 27% alpha helix as determined by circular dichroism studies. Both are sensitive to Pronase and resistant to trypsin, collagenase, and elastase. The lighter chain is stable to pepsin but the heavier can be partially degraded to a smaller polypeptide with a molecular weight similar to that of light chain. Amino acid analysis shows that the light chain contains 12 more tyrosine residues than does the heavy chain, suggesting that the light chain is not generated from the heavy chain. However, the two chains may have a common peptide region. Antiserum prepared against the heavier polypeptide can be completely absorbed by purified lighter polypeptide and vice versa indicating that both chains have some common antigenic determinants. Antibody against either chain can cross-react with the stratum corneum and keratohyalin granules in the epidermis of newborn rat as indicated by fluorescent microscopic observation. Similarly, this antibody also cross-reacts with the cell surface or the contents of spinous and granular cells, and very weakly with basal cells, indicating that the two proteins may be present in the lower strata as well as the stratum corneum.     

Histidine uptake in the epidermis of lizards and snakes in relation to the formation of the shedding complex

Mammalian epidermis utilizes histidine-rich proteins (filaggrins) to aggregate keratin filaments and form the stratum corneum. Little is known about the involvement of histidine-rich proteins during reptilian keratinization. The formation of the shedding complex in the epidermis of snakes and lizards, made of the clear and the oberhautchen layers, determines the cyclical epidermal sloughing. Differently from snakes, keratohyalin-like granules are present in the clear layer of lizards. The uptake of tritiated histidine into the epidermis of two lizards and one snake has been studied by autoradiography in sections at progressive post-injection periods. At 40 min and 1 hr post-injection keratohyalin-like granules were not or poorly labeled. At 3-22 hr post-injection most of the labeling was present over suprabasal cells destined to form the shedding complex, in keratohyalin-like granules of the clear layer, and in the forming a-layer but was low in the forming b-layer, and in superficial keratinized layers. The analysis of the shedding complex in the pad lamellae (a specialized scale used for climbing) of a gecko showed that the setae and the cytoplasm of clear cells among them are main sites of histidine uptake at 4 hr post-injection. In the snake most of the labeling at 4 hr post-injection was localized in the shedding complex along the boundary between the clear and oberhautchen layers. The present study suggests that, in the epidermis of lepidosaurian reptiles, the synthesis of a histidine-rich protein is involved in the formation of the shedding layer and, as in mammals, in a-keratinization.     

Normal human epidermal keratinocytes express in vitro specific molecular forms of (pro)filaggrin recognized by rheumatoid arthritis-associated antifilaggrin autoantibodies.

BACKGROUND: The so-called antikeratin antibodies and the antiperinuclear factor are the most specific serological markers of rheumatoid arthritis (RA). They were recently shown to be largely the same autoantibodies and to recognize human epidermal filaggrins and profilaggrin-related proteins of buccal epithelial cells (collectively referred to as (pro)filaggrin). MATERIALS AND METHODS: To further characterize the target antigens, we investigated their expression by normal human epidermal keratinocytes cultured in differentiating conditions, using immunofluorescence and immunoblotting with RA sera and three different monoclonal antibodies to (pro)filaggrin. RESULTS: On the cornified, stratified epithelial sheets obtained in vitro, RA sera with anti(pro)filaggrin autoantibodies (AFA) produced granular staining of the stratum granulosum and diffuse staining of the stratum corneum. The antigens recognized by RA sera strictly colocalized with (pro)filaggrin in keratohyalin granules. Following sequential extraction of the proteins from the epithelial sheets, the RA sera and the three monoclonal antibodies to (pro)filaggrin, recognized a series of low-salt-soluble molecules, including a neutral/acidic isoform of filaggrin and several proteins with sizes and pI intermediates between this isoform and profilaggrin. They also recognized urea-soluble high-molecular-weight profilaggrin-related molecules. CONCLUSIONS: These results show that in vitro epidermal keratinocytes express various molecular forms of (pro) filaggrin that bear epitopes targeted by AFA of RA sera, and that some of these are absent from epidermis. Moreover, these epitopes, which are present on the keratohyalin granules of buccal epithelial cells but not on those of epidermal cells, are present on the granules of the cultured keratinocytes. This work completes the molecular characterization of the proteins targeted by AFA.     

Immunohistochemical analysis of stratum corneum components in oral squamous epithelia

The generation of a stratum corneum in squamous epithelia involves marked changes in morphology and in the expression of cell products. We have examined the expression of some of the components involved in this process in oral squamous epithelia with different terminal differentiation patterns by use of immunofluorescent techniques. Involucrin and transglutaminase are involved in formation of cornified envelopes consistently seen in the stratum corneum. Both components were present in keratinized oral epithelia (palatal epithelium and hyperkeratinized buccal epithelium). The nonkeratinized normal buccal epithelium stained positive as well. Filaggrin, a protein derived from a precursor present in keratohyalin granules, is proposed to aggregate keratin filaments in the cornified layer. Although the staining differed markedly in quantity, this component was likewise detected in both keratinized and nonkeratinized epithelia. The staining patterns for different keratin polypeptides, however, showed qualitative differences between the different epithelia. Thus, it seems that the keratin composition shows differentiation-specific characteristics, whereas the presence of other important components needed to generate a stratum corneum is not as closely related to the terminal differentiation pattern of oral epithelia.     

Immunocytochemical localization of sulfhydryl oxidase in mammalian epidermis suggests that the enzyme cross‐links keratins in the granular and transitional corneous layers

The epidermis of representative mammalian species including humans has been examined for the presence of sulfhydryl oxidase, an enzyme likely involved in the oxidation of corneous proteins containing sulfhydryl groups in the epidermis. A database search indicates that the enzyme shares common sequences in numerous mammalian species so that an antibody against the human sulfhydryl oxidase 2 has been utilized on other species. The immunofluorescent study on the epidermis of the platypus (monotreme), red kangaroo (marsupials), hamster and human (placentals) reveals a prevalent labelling in the granular, transitional and lowermost part of the stratum corneous layer. The detailed ultrastructural immunogold study of the human epidermis reveals a diffuse and uneven labelling in the paler component of the composite keratohyalin granules or among keratin filaments of the transitional layer while the labelling disappears in the corneous layer. The study supports the hypothesis of the participation of the enzyme in the oxidative process that determines the formation of stable disulphide groups among keratins and other corneous proteins of the stratum corneum. This process gives rise to the resistant cell corneous envelope of keratinocytes in addition to the isopeptide bonds that derive from the catalytic action of epidermal transglutaminase on several corneous proteins.     

Local cutaneous water barrier in cold- and heat-acclimated pigeons (Columba livia) in relation to cutaneous water evaporation

The thermoregulatory function of the skin differs in adult cold-acclimated and heat-acclimated rock pigeons (Columba livia). In general, the cutaneous evaporative cooling mechanism is not activated by appropriate stimuli in cold-acclimated pigeons in contrast to heat-acclimated pigeons. We studied with electron microscopy whether the differences in the function of the skin are reflected in the structure of the epidermal water barrier of these two extreme acclimation states. The epidermis of cold-acclimated pigeons is attenuated, and the underlying dermis lacks any intimate vascularization. Both the extracellular and the intracellular domains in the stratum corneum contain organized lamellar lipids. At the stratum transitivum-stratum corneum interface, multigranular body secretion is indicated by the highly convoluted cell membranes and membraneous sacculae enclosing the multigranular bodies. Alternatively, multigranular bodies retain in the corneocytes, and the lipoid material originated from them is reprocessed to broad lamellae. The keratohyalin (KH) granules are spotlike and oriented as cortical bands beneath the plasma membrane. In heat-acclimated pigeons, the epidermis displays modified patches side by side with basic structural type of epidermis. The modified areas are characterized by hypertrophy and abundance of dermal capillaries adjacent to the hypertrophied patch. No lamellar lipids are discerned in the dilated extracellular space. The structure of multigranular bodies is abnormal, and the numbers of lipid droplets in the outer viable epidermis and stratum corneum are decreased. The transitional cells contain stellate KH granules, which form a network throughout the cell. It is concluded that cold-acclimated pigeons have a lamellar, extracellular water barrier, the cutaneous water evaporation is minimized, and heat is stored in the body core. Acclimation to heat leads to formation of structurally heterogeneous skin. The structurally modified skin patches show disruption of the barrier-forming machinery in the multigranular bodies and marked reorganization of fibrillar proteins and electron-dense KH masses in the transitional layer. Thus water barrier adjustments in cold- and heat-acclimated pigeons manifest the dynamic function of avian skin as a thermoregulatory organ.     

Tissue-specific expression of keratin proteins in human esophageal and epidermal epithelium and their cultured keratinocytes

In contrast to the simplified keratin content of bovine, rabbit, and rat esophageal epithelium (composed mainly of a 57 and 46 or 51 kD keratin, depending on the animal species), human esophageal epithelium contained a quantitatively different array of keratin proteins, ranging in molecular weight from 37 to 61 kD. The pattern of keratin proteins from human esophageal epithelium differed qualitatively and quantitatively from that of human epidermis. Human esophageal epithelium lacked the 63, 65, and 67 kD keratins characteristic of human epidermis, consistent with the absence of a granular layer and an anucleate stratum corneum. Moreover, human esophageal epithelium contained a distinctive 61 kD keratin protein which was either not present or present in only small amounts in human epidermis and variable amounts of a 37 kD keratin. Whereas the 56, 59, and 67 kD keratins were the most abundant keratins in human epidermis, the 52, 57, and 61 kD keratins predominated in human esophageal epithelium. During in vitro cultivation, both human epidermal and esophageal keratinocytes produce colonies which are stratified, but the morphologic appearance of these cultured epithelia differs. Only cultured human epidermal keratinocytes contain keratohyalin granules in the outermost layers and a prominent 67 kD keratin on immunoprecipitation. Otherwise the keratin contents appear similar. In conclusion, human esophageal epithelium exhibited intertissue and interspecies differences in the pattern of keratin proteins. During in vitro cultivation, human esophageal keratinocytes retained some aspects of their distinctive program of differentiation.     

Vitamin C enhances differentiation of a continuous keratinocyte cell line (REK) into epidermis with normal stratum corneum ultrastructure and functional permeability barrier

A continuous rat epidermal cell line (rat epidermal keratinocyte; REK) formed a morphologically well-organized epidermis in the absence of feeder cells when grown for 3 weeks on a collagen gel in culture inserts at an air-liquid interface, and developed a permeability barrier resembling that of human skin. By 2 weeks, an orthokeratinized epidermis evolved with the suprabasal layers exhibiting the differentiation markers keratin 10, involucrin, and filaggrin. Granular cells with keratohyalin granules and lamellar bodies, and corneocytes with cornified envelopes and tightly packed keratin filaments were present. Morphologically, vitamin C supplementation of the culture further enhanced the normal wavy pattern of the stratum corneum, the number of keratohyalin granules present, and the quantity and organization of intercellular lipid lamellae in the interstices of the stratum corneum. The morphological enhancements observed with vitamin C correlated with improved epidermal barrier function, as indicated by reduction of the permeation rates of tritiated corticosterone and mannitol, and transepidermal water loss, with values close to those of human skin. Moreover, filaggrin mRNA was increased by vitamin C, and western blots confirmed higher levels of profilaggrin and filaggrin, suggesting that vitamin C also influences keratinocyte differentiation in aspects other than the synthesis and organization of barrier lipids. The unique REK cell line in organotypic culture thus provides an easily maintained and reproducible model for studies on epidermal differentiation and transepidermal permeation.     

The binding of epidermolytic toxin from Staphylococcus aureus to mouse epidermal tissue

Summary Fluorescein-labelled epidermolytic toxin (FTC-toxin) ofStaphylococcus aureus and ferritin—toxin conjugate have been prepared and purified. FTC-toxin bound selectively to cryostat and resin-impregnated sections of neonatal mouse skin. Binding was localized at the keratohyalin granules and in the stratum corneum. In an epidermal cell (granular, spinous and basal) preparation, only keratohyalin granules of the granular cells bound FTC-toxin. Ferritin—toxin conjugate bound to skin sections at the same two sites as FTC-toxin and was competitive with the binding of free toxin. Keratohyalin granules in unstained sections had a novel patched appearance under the electron microscope, and the ferritin—toxin conjugate bound preferentially to the electron-lucent areas. In the stratum corneum it was shown by quantitative estimation that the target density decreased as the surface of the tissue was approached.     

Staining of keratin and keratohyalin with the reactive dye levafix red violet E-2BL.

Demonstration of keratin in Zenker-fixed skin and in tissues stored in formalin can be difficult because such material is unsuitable for histochemical studies. A reactive dye, Levafix red violet E-2BL, proved useful for demonstration of keratohyalin and some types of keratin. Formalin-, Zenker- and methacarn-fixed sections were pretreated with alkaline alcohol, stained one hour at 60 C in an aqueous solution containing 0.25% Levafix red violet E-2BL plus 0.25% NaCl, rinsed in buffer solution pH 9, dehydrated and mounted. Keratohyalin granules and stratum corneum were colored red violet; hair and tonofibrils remained unstained. In sections prestained with Mayer's acid hemalum, keratohyalin was dark blue. Sulfonated monoazo dyes without reactive groups colored no tissue structures under the conditions of this technic; apparently, Levafix red violet E-2BL is bound via its reactive group. Polarization microscopic studies suggest binding of Levafix red violet E-2BL by an amorphous matrix of keratin. Correlations with chemical data indicate that the staining patterns parallel the distribution of proteins formed in the stratum granulosum.