Affinity purification of in vivo assembled whirlin-associated protein complexes from the zebrafish retina.

Mutations in WHRN lead to Usher syndrome type 2d or to non-syndromic hearing impairment. The WHRN-encoded gene product whirlin directly interacts with the intracellular regions of the other two Usher syndrome type 2-associated proteins, usherin and ADGRV1. In photoreceptor cells, this protein complex constitutes fibrous links between the periciliary membrane and the connecting cilium. However, the molecular mechanism(s) of retinal degeneration due to compromised formation and function of the USH2-associated protein complex remains elusive. To unravel this pathogenic mechanism, we isolated and characterized whirlin-associated protein complexes from zebrafish photoreceptor cells. We generated transgenic zebrafish that express Strep/FLAG-tagged Whrna, a zebrafish ortholog of human whirlin, under the control of a photoreceptor-specific promoter. Affinity purification of Strep/FLAG-tagged Whrna and associated proteins from adult transgenic zebrafish retinas followed by mass spectrometry identified 19 novel candidate associated proteins. Pull down experiments and dedicated yeast two-hybrid assays confirmed the association of Whrna with 7 of the co-purified proteins. Several of the co-purified proteins are part of the synaptic proteome, which indicates a role for whirlin in the photoreceptor synapse. Future studies will elucidate which of the newly identified protein-protein interactions contribute to the development of the retinal phenotype observed in USH2d patients. SIGNIFICANCE: Since protein-protein interactions identified using targeted in vitro studies do not always recapitulate interactions that are functionally relevant in vivo, we established a transgenic zebrafish line that stably expresses a Strep/FLAG-tagged ortholog of human whirlin (SF-Whrna) in photoreceptor cells. Affinity purification of in vivo-assembled SF-Whrna-associated protein complexes from retinal lysates followed by mass spectrometry, identified 19 novel candidate interaction partners, many of which are enriched in the synaptic proteome. Two human orthologs of the identified candidate interaction partners, FRMPD4 and Kir2.3, were validated as direct interaction partners of human whirlin using a yeast two-hybrid assay. The strong connection of whirlin with postsynaptic density proteins was not identified in previous in vitro protein-protein interaction assays, presumably due to the absence of a biologically relevant context. Isolation and identification of in vivo-assembled whirlin-associated protein complexes from the tissue of interest is therefore a powerful methodology to obtain novel insight into tissue specific protein-protein interactions and has the potential to improve significantly our understanding of the function of whirlin and the molecular pathogenesis underlying Usher syndrome type 2.

Vestibular function and temporal bone imaging in DFNB1.

DFNB1 is the most prevalent type of hereditary hearing impairment known nowadays and the audiometric phenotype is very heterogeneous. There is, however, no consensus in literature on vestibular and imaging characteristics. Vestibular function and imaging results of 44 DFNB1 patients were evaluated in this retrospective study. All patients displayed a response during rotational velocity step testing. In 65% of the cases, the caloric results were within normal range bilaterally. The video head impulse test was normal in all patients. In 34.4% of the CT scans one or more temporal bone anomalies were found. The various anomalies found, were present in small numbers and none seemed convincingly linked to a specific DFNB1genotype. The group of DFNB1 patients presented here is the largest thus far evaluated for their vestibular function. From this study, it can be assumed that DFNB1 is not associated with vestibular dysfunction or specific temporal bone anomalies.

Developmental aspects of the rat endolymphatic sac and functional implications.

The purpose of this study was to determine specific characteristics of endolymphatic sac (ES) cells of the developing rat that are considered to be involved in endolymph homeostasis. Because intermediate filament proteins (IFPs) are regarded as markers of cell differentiation and basal lamina proteins (BLPs) are essential in cell<=>matrix interactions, we determined the presence of IFPs [cytokeratins (CKs) and vimentin] and BLPs [collagen IV, heparan sulphate proteoglycan (HSPG) and laminin] at different developmental stages before and after birth. In addition, we studied the expression of two enzymes of oxidative metabolism: cytochrome oxidase and succinate dehydrogenase. The presence of CKs 8, 18 and 19 in all epithelial cells of the ES during the embryonic stage is characteristic of simple (glandular) epithelial cells. Interestingly, a distinct population of these cells shows additional expression of CK 7, which is a feature of secretory cells. These CK 7-positive cells also contain a high concentration of oxidative enzymes and are rich in mitochondria, indicating that they are light cells. It is suggested that light cells possess specific energy-requiring transport capabilities. Loss of CK 19 expression in the distal part and in a large region of the intermediate part of the ES implies that these cells do not differentiate any further and acquire the capacity to proliferate. Furthermore, prominent co-expression of vimentin with the CKs in the distal part of the ES may confer viscoelastic properties on this epithelium. This may facilitate expansion and thus enable cushioning of pressure fluctuations. Finally, the early prominent occurrence of HSPG in the basal lamina of the ES enables transport of ions. In this light our recent observations of early functioning NaK-ATPases in certain ES cells are interesting.

Occurrence of NaK-ATPase isoforms during rat inner ear development and functional implications.

This study examined the presence of NaK-ATPase isoforms in the developing inner ear of the rat and studied the importance of functional subunit combinations in endolymph homeostasis. The findings were: (a) the combination alpha 1 beta 1 is found in epithelial, mesenchymal, and neural inner ear cells with an early starting expression 14 days postconception (dpc) in some endolymphatic sac cells; (b) from 1 day after birth (dab) expression of alpha 1 beta 2 is observed in marginal cells, vestibular dark cells, and certain vestibular nonsensory cells; (c) a transient expression of alpha 2 beta 1 is found in suprastrial fibrocytes and spiral ligament fibrocytes type II between 10 and 15 dab; (d) starting at 16 dpc the combination alpha 3 beta 1 is uniquely expressed in inner ear neural cells (as in other neural tissues). In conclusion, during development a switch from alpha 2 beta 1 towards alpha 1 beta 1 is observed in suprastrial fibrocytes and in spiral ligament fibrocytes type II. Thus, according to the biochemical characteristics of these combinations, a switch towards a NaK-ATPase with higher capacity takes place. In addition, prominent expression of the alpha 1 beta 2 combination in predominantly K+ ion transporting marginal and dark cells is in accordance with the characteristic of this combination and thus with the presumed function of these cells as important K+ suppliers for the endolymph. We believe this combination in certain vestibular nonsensory cells to be involved in K+ sensing. Early expression of the alpha 1 beta 1 combination in the endolymphatic sac, prior to that in the other parts of the inner ear, suggests that this structure may be involved to some extent in the development of the vestibulum and cochlea.

Middle ear effusions and structure of the tympanic membrane.

OBJECTIVE: To study the effect of various middle ear effusions on the structure of the lamina propria of the tympanic membrane. METHODS: Sterile and infective middle ear effusions were induced by obstruction of the eustachian tube in specific pathogen-free (SPF) rats and in rats with upper airway infections (URI), respectively. The condition of the tympanic membrane was monitored at regular intervals. After varying survival times, the animals were killed and the tympanic membranes processed for light and electron microscopy. RESULTS: Sterile effusions always resulted in tympanosclerotic lesions. These lesions did not develop in the presence of primary-infected effusions. These effusions had a severe destructive effect on the lamina propria, followed by fibrosis. Generally, secondary infection did not markedly affect preexisting tympanosclerotic lesions. Moreover, calcification disappeared when re-aeration of the middle ear occurred, but the abnormal collagen depositions persisted. CONCLUSIONS: Both sterile and infective effusions result in comprehensive irreversible changes in the lamina propria of the pars tensa. The development of tympanosclerosis is confined to sterile effusions. Mechanical injury and compromised vascularization of the lamina propria are likely to be important etiological factors in the development of tympanosclerosis.

Improved purification of water and waste-water with low-cost dead-end-ultrafiltration.

One aspect of the increasing contamination of water sources is sewage effluent. In some countries it is generally disinfected prior to discharge to protect downstream communities, which use the water for drinking and recreation. However, serious questions have been raised regarding the efficacy of conventional treatments to remove or destroy viruses, and regarding the generation of harmful byproducts by sewage chlorination. A safe and reliable solution is the use of ultrafiltration for the purification of wastewater, as the ultrafiltration membranes form an absolute barrier for bacteria and viruses including colloids and macromolecules. However, this application also demands the use of open channel module systems that can be cleaned with high efficiency with regard to scaling, fouling and especially biofouling. The flat membrane module systems using selected membranes and a special plant design can meet these and further requirements expected in this application, including easy handling, low energy consumption and optimised operation costs. Technical details of the module system, case studies and cost aspects are presented.

Osteochondritis dissecans of the patellofemoral joint.

Osteochondritis dissecans of the patellofemoral joint is an uncommon condition that may be the cause of anterior knee pain or crepitus. We present the clinical features of 37 patients with osteochondritis dissecans lesions of the patellofemoral joint (24 on the patella, 13 on the trochlear groove), including two patients with medial trochlear groove lesions, which have not, to our knowledge, been previously reported. The osteochondral lesions involved the convex articular surfaces. The median age of patients when first examined was 15 years, and 54% of patients had open epiphyses. These lesions were more common in male patients than in female patients (four-to-one ratio). Osteochondritis dissecans of the patellofemoral joint can be overlooked unless quality radiographs are viewed with care and, at arthroscopy, both the patella and trochlear groove are assessed. Treatment depends on the symptoms, site, and nature of the lesion and the patient's age. Nonoperative management includes patellar taping and vastus medialis obliquus muscle exercises. Operative intervention is indicated for patients with mechanical symptoms and includes arthroscopy, consisting of chondroplasty and removal of loose bodies, and lateral retinacular release. In this study treatment generally improved the symptoms, but patients with articular cartilage loss had persistent patellofemoral crepitus and discomfort.

Nature of the tympanic membrane insertion into the tympanic bone of the rat.

The nature of the insertion of the tympanic membrane into the tympanic bone was studied in the rat during the developmental period ranging from 18 days post conception (dpc) to 40 days after birth (dab). Techniques applied were light microscopy, electron microscopy and immunohistochemistry with antibodies to cytoskeletal proteins: vimentin, desmin and alpha-smooth muscle actin (sma) as fibroblast differentiation markers. It was established that the cartilaginous annulus of the pars tensa was connected to the tympanic bone by an interface of specialised connective tissue. Both the fibrocartilage and the interface were derived from the embryonal mesenchyme between the tympanic ring and meatal plate. Electron microscopy showed that the interface was composed of two types of fibroblast. The majority of these cells were myofibroblasts, which were interconnected by junctions and had intimate contact with the collagenous fibres. A small number were identified as genuine fibroblasts. Cytoskeletal characterisation revealed the presence of three types of cell: V cells which expressed vimentin, VA cells which expressed vimentin and alpha-sma and VAD cells which expressed vimentin, alpha-sma and desmin. The myofibroblasts expressed antigens of both smooth muscle cells (alpha-sma, desmin) and connective tissue cells (vimentin). It is suggested that the pars tensa is connected to the tympanic bone by a network of contractile cells and fibres. Contraction will move the membrane in an outward direction and antagonise the inward retraction by the tensor tympani.

Development of the tubotympanum in the rat.

OBJECTIVE: To investigate the relationship between the anatomical maturation of the middle ear and that of the eustachian tube and paratubal muscles in the rat. DESIGN: Wistar rats ranging from gestational day 12 to postnatal day 40 were used. METHODS: Tissue specimens were examined with routine light microscopy and electron microscopy. Epithelial differentiation was studied immunohistochemically with antibodies to different cytokeratins. RESULTS: The epithelial lining of the tubotympanum showed differentiation-related cytokeratin expression throughout the whole developmental period. The mucociliary epithelium reached mature features around birth. A dorsal extension and its framing cartilage started forming around 5 days after birth. This extension became lined by stratified nonciliated epithelium and attained maturity around 10 days after birth concurrently with the attachment of the dilatory muscles. This process was immediately followed by aeration of the middle ear cavity. CONCLUSIONS: The continuous expression of cytokeratins demonstrates that the epithelial lining of the tubotympanum is only derived from the embryonal endoderm. Furthermore, this study demonstrates that the eustachian tube shows a two-stage postnatal development. First, the mucociliary system matures, providing protection/clearance when the animal starts respiration and swallowing. Subsequently, the dorsal part attains maturity. The features of the epithelial lining of the dorsal part of the eustachian tube and the coincidence of the maturation of this part with the attachment of the dilating muscle fibers and the aeration of the middle ear indicates that this part provides ventilation. These findings support the authors' hypothesis that different parts of the eustachian tube serve different purposes: clearance, protection and ventilation.

Keratinocyte differentiation in acquired cholesteatoma and perforated tympanic membranes.

OBJECTIVE: To evaluate the type of differentiation of keratinocytes of acquired cholesteatoma and its significance for cholesteatoma invasiveness. DESIGN: Forty acquired cholesteatomas and 10 tympanic membranes with persisting perforations were snap frozen and processed for immunohistochemical studies. Cytokeratin antibodies that represented all subgroups and antibodies that were directed against collagen components of the basal lamina were applied. Expression of these constituents was scored by using light microscopy. RESULTS: The phenotype of the matrix was generally characterized by an extension of expression of basal cell cytokeratin 14 and hyperproliferation-associated cytokeratins 6, 16, and 17 into the suprabasal cell layers, while the expression of keratinization marker cytokeratin 10 was down-regulated. These features varied greatly at different sites of the matrix and were most marked at the advancing front of the cholesteatoma. A comparable expression pattern, but less pronounced, was observed at the epidermal front of the mucocutaneous junction of the tympanic membrane perforations. This phenomenon was invariably associated with a mononuclear cell infiltrate in the dermis at both junctions. The basal lamina was always intact. CONCLUSIONS: Acquired cholesteatomas show hyperproliferative features. There is a striking similarity between the pronounced expression of this phenotype and the associated inflammation at the mucocutaneous junctions of cholesteatomas and tympanic membrane perforations and those that are observed after epidermal injury. This indicates that epidermis and middle ear epithelium do not form stable junctions and the front can be considered to be a persisting epidermal defect. This involves the permanent presence of "activated keratinocytes" in the junction area that will lead to proliferation and migration, when additional triggers are present.

Macrophage subpopulations and RPE elimination in the pathogenesis of experimental autoimmune pigment epithelial protein-induced uveitis (EAPU).

Experimental autoimmune pigment epithelial protein-induced uveitis (EAPU) is a new type of disease that destroys the retinal pigment epithelium (RPE), and exhibits a hitherto unknown form of progressive chorioretinal dystrophy in which neuroretinal inflammatory foci are absent. The present study was aimed at studying the expression of adhesion molecules, and the kinetics of the appearance of the main types of macrophages and other intraocular immunocompetent cell populations in the various stages of this disease. EAPU was evoked in Lewis rats by immunization with the membrane protein from bovine RPE cells containing PEP-65 as main constituent. In the uvea, increased expression of intercellular adhesion molecule-1, of class II major histocompatibility complex antigen, and of ED2 macrophage reactivity were observed closely before the onset of EAPU. Expression of these reactivities was also slightly elevated by injections of the applied adjuvants alone. The onset of EAPU was mainly characterized by initial uveal infiltrations of ED1+ macrophages and a minor population of CD4 T cells, and an increase in ED3, ED7 and perivascular ED2 reactive macrophages. This was followed by the development of focal accumulations of ED1+ cells at both sides of the Bruch's membrane-RPE layer (Dálen-Fuchs nodules) which was permeated and disintegrated at these sites. The outer choroidal layer, the anterior iridal surface, and the base of the ciliary body more frequently contained active inflammatory cells than the other uveal areas. Lymphoid cells were found scattered through the uvea, aqueous and vitreous. The sites of increased activity of ED2+ and ED3+ cells in the uvea were rather similar to those of ED1 macrophages in the various stages of EAPU. Starting from multiple foci, the process of the formation of plaque-shaped cell accumulations in severe EAPU progressed along the RPE and exhibited a chronic character. The results of this study show that ED1+, ED2+, ED3+ and ED7+ subpopulations of macrophages are actively involved in an immunopathological process in which the RPE is the target. The thickening of the plaque-shaped cell accumulations stops if the integrity of all RPE cells at that site has been affected. We postulate that this is the result of antigen elimination while additional influence of the abrogation of RPE cytokine production is presumed.

Epidermal differentiation in the human external auditory meatus.

The differentiation of epidermis in the various parts of the human ear canal was documented on the basis of cytokeratin (Ck) expression patterns. Immunohistochemistry was performed on cryostat sections of normal meatal skin using a comprehensive panel of monospecific Ck antibodies representing the main lines of epithelial differentiation. The epidermis of the cartilaginous part showed a Ck profile characteristic of normal skin type differentiation. The deep meatal skin, including the tympanic membrane, showed a peculiar type of differentiation: in addition to epidermal Cks, hyperproliferation-associated Cks 6, 16, and 17 were expressed in the suprabasal cells, while the simple epithelia cell marker Ck 19 was found in the basal cells. The presence of hyperproliferative Cks in the deep meatal skin could only partly be related to areas of proliferative activity. Keratinocytes, which express markers of hyperproliferation, are migratory. Therefore, their presence in the meatal skin is likely to be related to the peculiar pattern of keratinocyte migration, the purpose of which is to keep the meatus free from desquamation products.

Squamous metaplasia of the middle ear epithelium.

This study deals with the expression of cytokeratins (Cks) in squamous cell metaplastic lesions in rat and human middle ear. In rats, squamous metaplastic lesions could be induced during chronic otitis media. The histological features of these lesions were similar to those observed in the human middle ear. Immunohistochemistry revealed that squamous cell metaplasia in both rat and human middle ear is characterised by a loss of simple epithelial cell related Cks and the appearance of Cks characteristic of stratified and cornifying epithelia. This indicates a true change in the differentiation of the middle ear epithelium. It is concluded that the Ck profile of the cholesteatoma matrix cannot be used as a variable to decide whether the origin of cholesteatomas is epidermal or metaplastic. This rat model is suitable for studying squamous cell metaplasia in relation to cholesteatoma genesis.

Distribution and features of melanocytes during inner ear development in pigmented and albino rats.

In this developmental study, the distribution and features of melanocytes in the inner ear of pigmented and albino rats was investigated with the use of an antibody, which specifically reacts with a melanocyte differentiation antigen present in the membranes of (pre)melanosomes. Melanocyte precursors could be traced from 13 days post conception onwards and the course was followed to their targets in the inner ear. Melanocytes which settle in the modiolus appeared to reach their target along another pathway than strial and vestibular melanocytes. No difference was observed in the melanocyte distribution between pigmented and albino rats. The integration of melanocytes into the stria vascularis was associated with an increased rate of melanosome production in both strains, but in the albinos far fewer melanosomes were produced. After the stria had reached maturity, melanosome production was arrested and melanosomes were subject to lysosomal digestion. In the stria of the pigmented rats, cells with aggregations of disintegrating melanosomes appeared and persisted into adulthood. In the adult, the majority of the intermediate cells contained only a few scattered melanosomes, while melanosomes could only rarely be detected in the albinos. These observations indicate that there is a close relationship between melanosome production and the process of interdigitation of melanocytes with the marginal cells. It seems unlikely that melanosomes or melanin make any important contribution to the function of the adult stria vascularis. Outside the stria, the features of melanocytes in both strains were similar to skin melanocytes.

Growth and differentiation of meatal skin grafts in the middle ear of the rat.

OBJECTIVE: To determine the behavior of epidermal cells after transplantation in the middle ear. DESIGN: In a rat model, full-thickness meatal skin grafts were transplanted into the middle ear and studied morphologically and immunohistochemically with the use of antibodies directed against different cytokeratin (Ck) polypeptides, which are markers of different types of epithelial cell differentiation. RESULTS: The grafts had either transformed into epithelial cysts or had become integrated into the middle ear epithelium. The epithelium of the integrated grafts showed gradual transition into the epithelium of the middle ear. A clear distinction between epidermal cells and middle ear epithelium could be made only on the basis of their Ck profiles. The Ck profiles of the grafts revealed a decrease in the expression of epidermal Cks, while nonepidermal Cks became expressed. These changes can be ascribed to replacement of the dermal mesenchyma by mesenchyma from the middle ear. In two ears with superimposed infection, the graft epithelium showed expansive growth. CONCLUSIONS: Meatal epidermis is well tolerated in the middle ear, but superimposed infection can induce expansive growth. These findings favor the concept that the progressive growth of cholesteatoma is related to the presence of inflammatory processes.

Developmentally-regulated coexpression of vimentin and cytokeratins in the rat inner ear.

In the present study the expression of vimentin-type intermediate filament proteins and cytokeratins was studied immunohistochemically in the rat inner ear from 12 days postconception up to 40 days after birth. With the use of a broad spectrum monoclonal antibody, cytokeratin expression was found to be present in the whole epithelial lining except for the sensory cells, throughout all the developmental stages examined. Vimentin was detected in the mesenchymal cells, the mesenchyme-derived tissues and the intermediate cells of the stria vascularis, confirming their origin from melanocyte precursor cells. In addition, the coexpression of vimentin and cytokeratins in the epithelial lining of the membranous inner ear was found to be developmentally regulated. During the final stages of differentiation, vimentin expression disappeared from the majority of the cell types. In the mature cochlea the coexpression of vimentin and cytokeratins was still found in the supporting cells of the organ of Corti, in the cells of Claudius and in external sulcus cells. As far as we could conclude from this study, the sensory cells showed only vimentin expression but not cytokeratin expression. A possible relationship between vimentin expression in adult epithelial cells of the inner ear and a specialised function of these cells is discussed.

Expression of intermediate filament proteins in the mature inner ear of the rat and guinea pig.

The expression of intermediate filament proteins was studied in the mature inner ear of the rat and guinea pig, using a panel of polyclonal and monoclonal antibodies directed against cytokeratins, desmin, neurofilament proteins and glial fibrillary acidic protein (GFAP). The epithelial lining of the endolymphatic space displayed a complex expression pattern of cytokeratin filament proteins, suggesting greater cell diversity than was known sofar from morphological studies. The cytokeratin antibodies when applied to the inner ear tissues revealed the presence of only cytokeratin polypeptides which are typical of simple epithelia (i.e. nos. 7, 8, 18, and 19). Profound differences in cytokeratin expression patterns were, however, found in the various cell types of both the cochlear and vestibular partition. Remarkably, the sensory cells appeared to be devoid of both cytokeratins and neurofilament proteins. Staining with a 200 kDa neurofilament antibody displayed the presence of different populations of ganglion cells in the spiral ganglion and the vestibular ganglion. There was no reaction with antibodies directed against desmin and GFAP. The great resemblance of the intermediate filament protein expression patterns in the inner ear of the rat and guinea pig indicates a close similarity between the different epitopes.

Expression of cytokeratin polypeptides during development of the rat inner ear.

The expression of cytokeratin polypeptides in the different epithelia of the developing inner ear of the rat from 12 days post conception to 20 days after birth was analysed immunohistochemically, using a panel of monoclonal antibodies. Throughout the development of the complex epithelial lining of the inner ear originating from the otocyst epithelium, only cytokeratins which are typical of simple epithelia were expressed. Cytokeratins 8, 18, and 19 were detectable shortly after the formation of the otocyst from the ectoderm (12 dpc), whereas cytokeratin 7 expression was delayed and first appeared in the vestibular portion and subsequently in the developing cochlear duct. During the development of the different types of specialized cells, differentiation-dependent modulation of the cytokeratin expression patterns was observed. In the mature inner ear, the specialized cell types displayed a function-related cytokeratin expression profile, both in the cochlear and vestibular portion. Cytokeratin expression in the flat epithelium of the vestibular portion suggests a more complex composition of this epithelium than has been established from routine morphology. Remarkably, the cochlear sensory cells were apparently devoid of cytokeratins, but no final conclusion could be drawn on the presence of cytokeratins in the sensory cells of the vestibular portion, because of the difficulty to delineate the cell borders between sensory cells and supporting cells.

Immunohistochemical study of cytokeratin expression in normal and pathologic middle ear mucosa of the rat.

The expression of cytokeratins in the epithelium of the middle ear and external auditory meatus of the rat was studied on cryosections of ethylenediaminetetraacetic acid-decalcified specimens by use of a panel of monoclonal antibodies. The normal middle ear epithelium revealed a complex cytokeratin profile, including regional differences. The induction of sterile middle ear effusions resulted in increased cytokeratin expression. Infective effusions were accompanied by both quantitative and qualitative changes in the cytokeratin expression patterns. The differences observed between the cytokeratin profiles of external meatal skin and those of middle ear epithelium may form a useful tool for research into cholesteatoma development.

Effect of EDTA on cytokeratin detection in the inner ear.

Immunohistochemical studies on the epithelium of the adult inner ear are difficult to perform without decalcification of the bony capsule. In this study, we examined the effect of decalcifying agents on the immunoreactivity of various cytokeratin antigens in the cochlear duct epithelium of 2-day-old rats, allowing the comparison of fresh and decalcified specimens. Decalcification of unfixed tissue in a solution containing EDTA or EGTA and polyvinylpyrrolidone, at pH 7.4 and 4 degrees C for a maximum period of 2 days, not only preserved the antigen epitopes but even enhanced the staining intensities in comparison with fresh specimens. This enhancement effect, caused by chelating agents and found to be blocked by prior fixation with acetone, is suggested to be caused by unmasking of the antigenic epitopes.