Human specific anti-type IV collagen monoclonal antibodies, characterization and immunohistochemical application

This paper describes two new monoclonal antibodies reactive with human specific type IV collagen epitopes in frozen as well as routinely fixed and processed tissue sections. The antibodies (1042 and 1043) were raised against human placental type IV collagen and were shown by immunoblotting and ELISA tests to react exclusively with type IV collagen determinants. Extensive immunohistochemical survey studies on panels of tissues from various species, using unfixed cryostat sections, demonstrated that antibody 1042 reacted only with human type IV collagen whereas antibody 1043 in addition reacted with rabbit type IV collagen. All tissues showed homogeneous staining of the basement membrane, indicating that the detected epitopes did not show organ-specific distribution. Tissue processing protocols for using these monoclonal antibodies on routinely processed paraffin embedded tissues were developed. It was found that whereas polyclonal anti-type IV collage antisera required pepsin digestion, our monoclonal antibodies required pronase or papain digestion to restore type IV collagen immunoreactivity in paraffin sections. It is concluded that these monoclonal anti-type IV collagen antibodies detect species specific epitopes which can be detected in routinely processed paraffin embedded tissues after appropriate enzyme pretreatment.     

Human specific anti-type IV collagen monoclonal antibodies,characterization and immunohistochemical application

Summary This paper describes two new monoclonal antibodies reactive with human specific type IV collagen epitopes in frozen as well as routinely fixed and processed tissue sections. The antibodies (1042 and 1043) were raised against human placental type IV collagen and were shown by immunoblotting and ELISA tests to react exclusively with type IV collagen determinants. Extensive immunohistochemical survey studies on panels of tissues from various species, using unfixed cryosat sections, demonstrated that antibody 1042 reacted only with human type IV collagen whereas antibody 1043 in addition reacted with rabbit type IV collagen. All tissues showed homogeneous staining of the basement membrane, indicating that the detected epitopes did not show organ-specific distribution.Tissue processing protocols for using these monoclonal antibodies on routinely processed paraffin embedded tissues were developed. It was found that whereas polyclonal antitype IV collagen antisera required pepsin digestion, our monoclonal antibodies required pronase or papain digestion to restore type IV collagen immunoreactivity in paraffin sections.It is concluded that these monoclonal anti-type IV collagen antibodies detect species specific epitopes which can be detected in routinely processed paraffin embedded tissues after appropriate enzyme pretreatment.     

pH-dependent function, purification, and intracellular location of a major collagen-binding glycoprotein

A major collagen-binding heat shock protein of molecular mass 47,000 D was found to bind to collagen by a pH-dependent interaction; binding was abolished at pH 6.3. Native 47-kD protein could therefore be purified from chick embryo homogenates in milligram quantities by gelatin-affinity chromatography and gentle acidic elution. Rat monoclonal and rabbit polyclonal antibodies were generated against the purified 47-kD protein. Immunofluorescence microscopy of cultured chick embryo fibroblasts with these antibodies revealed bright, granular perinuclear staining as well as a weaker reticular network structure towards the cell periphery, suggesting that this protein was located in the endoplasmic reticulum. No immunofluorescence staining was detected on the cell surface. Double-staining experiments with these antibodies and fluorescently labeled wheat-germ agglutinin suggested that the 47-kD protein was absent from the Golgi apparatus. Localization of the 47-kD protein in the endoplasmic reticulum but not in the Golgi complex was confirmed by immunoelectron microscopy. In vivo localization studies using immunohistochemistry of cryostat sections of chick liver revealed that the 47-kD protein was present in fibrocytes, Kupffer cells, and smooth muscle cells. It was absent from hepatocytes and the epithelia of bile ducts or sinusoidal endothelium. This major transformation- and heat shock-regulated glycoprotein is thus localized intracellularly, is expressed in only certain cells, and functions in a pH-regulated manner. These findings suggest that this glycoprotein is not likely to be a general cell-surface collagen receptor, but may instead play roles in intracellular protein processing or translocation.     

Distribution of type IV collagen, laminin, and fibronectin during maxillary process formation in the chick embryo

The presence and distribution of laminin, type IV collagen, and fibronectin were analyzed in the facial primordia and developing primary palates of chick embryos from stages of development corresponding to maxillary process formation and primary palate closure. Frozen sections through the maxillary process and roof of the stomodeum were prepared for indirect immunofluorescence employing a biotin-avidin system using monoclonal antibodies against laminin, type IV collagen, and fibronectin. Light microscopic examination of sections stained with antibodies against type IV collagen revealed a much stronger fluorescent signal in the roof of the stomodeum than in the maxillary process at all stages examined. Regional differences in signal intensity and staining patterns were noted within the maxillary process; for example, the lateral surface of the maxillary process displayed a much less intense signal at most stages examined than the inferior and medial surfaces. The signal from sections of the maxillary process stained with laminin was much stronger than the signal from the same tissues stained with collagen. Regional differences in signal intensity within the maxillary process were minimal in sections stained with antibodies to laminin, in contrast to the differences seen in sections stained with antibodies to type IV collagen. Differences in signal intensity between the maxillary process and the roof of the stomodeum with laminin were slight. Sections stained with antibody to fibronectin displayed intense staining throughout the mesenchyme in both the maxillary process and the roof of the stomodeum. From comparison of the data of type IV collagen and laminin, the following hypothesis is proposed. In structures which undergo rapid change in form, such as the facial primordia, collagen distribution and/or organization is altered to a much greater extent than laminin, which is more uniformly distributed and which may be required for structural support of other developmentally regulated macromolecules. Where tissue morphology must be maintained, such as the roof of the stomodeum, the concentration and organization of type IV collagen is maintained in a manner that confers stability to these regions.     

Study of differential collagen synthesis during development of the chick embryo by immunofluorescence. I. Preparation of collagen type I and type II specific antibodies and their application to early stages of the chick embryo.

The aim of this work was to prepare specific antibodies against skin and bone collagen (type I) and cartilage collagen (type II) for the study of differential collagen synthesis during development of the chick embryo by immunofluorescence. Antibodies against native type I collagen from chick cranial bone, and native pepsin-extracted type II collagen from chick sternal cartilage were raised in rabbits, rats, and guinea pigs. The antibodies, purified by cross-absorption on the heterologous collagen type, followed by absorption and elution from the homologous collagen type, were specific according to passive hemagglutination tests and indirect immunofluorescence staining of chick bone and cartilage tissues. Antibodies specific to type I collagen labeled bone trabeculae from tibia and perichondrium from sternal cartilage. Antibodies specific to type II collagen stained chondrocytes of sternal and epiphyseal cartilage, whereas fluorescence with intercellular cartilage collagen was obtained only after treatment with hyaluronidase. Applying type II collagen antibodies to sections of chick embryos, the earliest cartilage collagen found was in the notochord, at stage 15, followed by vertebral collagen secreted by sclerotome cells adjacent to the notochord from stage 25 onwards. Type I collagen was found in the dermatomal myotomal plate and presumptive dermis at stage 17, in limb mesenchyme at stage 24, and in the perichondrium of tibiae at stage 31.     

Freeze-drying of bone tissue: immunocytochemistry and enzyme histochemistry on paraffin embedded and low-temperature resin embedded specimens

A simple protocol of tissue preparation was sought, which would enable marker enzymes of bone cells and extracellular matrix antigens to be localized in the same tissue section with high optical resolution. For this purpose, snap-frozen samples of rat fetal skeletal tissues were dried in a FDU 010 freeze-drying unit (Balzers) for 8–12 h at –50 to –40°C and 0.02 bar. Freeze-dried tissues were either vacuum-infiltrated at 45°C and embedded undemineralized in Paraplast, or vacuum-infiltrated overnight at 4°C and embedded undemineralized in glycol methacrylate. These procedures enabled enzyme cytochemistry for alkaline phosphatase and tartrate-resistant acid phosphatase, and immunocytochemical staining for collagen types I, III, and laminin to be performed on the same sections. No pretreatment of the sections was necessary to reveal collagen antigenicity. This study reveals the possibility of complementing immunocytochemical studies of extracellular matrix with enzyme cytochemistry and, above all, with the excellent tissue preservation and high resolution afforded by plastic embedding.     

Immunocytochemical analysis of cytocentrifuged fine needle aspirates. A study based on lung tumors aspirated in vitro

The value of Cytospin preparations of fine needle aspiration (FNA) biopsy material for immunocytochemical analysis was investigated using aspirates obtained from 23 resected human lung tumors. The results were compared with those on cryostat sections from the same tumors. The Cytospin preparations of the FNA biopsies gave the best immunostaining reactions and enabled a comprehensive range of monoclonal antibodies (MAbs) to be utilized. The quality of the Cytospin immunostaining compared favorably with that on cryostat sections of the same tumors and generally yielded a similar immunophenotype. However, the Cytospin preparations were not suitable for staining with MAb Ki67, which detects an antigen associated with cellular proliferation. With Ki67, conventionally prepared smears were much superior and enabled an assessment of tumor growth fraction that concurred with the growth fraction calculated from cryostat sections in most cases.     

Localization of different types of collagen (I, III, IV, V) in the connective tissue of the popliteal artery and skeletal muscle of man (immunoelectronmicroscopic analysis)

By means of immunoperoxidase and immunoferritin techniques collagen of the I, III, IV and V types has been revealed in cryostat sections of the popliteal artery and in the musculus quadriceps of the femur. Areas of the vascular wall without any macroscopical signs of lesions have been investigated. They have been obtained from amputated extremities of young persons (17-22 years old), and muscle pieces have been taken during operations performed in the knee joint. After certain immunocytochemical procedures the cryostat slices are embedded in mixture of epon 812 and araldit, non-contrasted ultrathin slices are examined in the electron microscope JEM 100CX. Collagen of the I and III types is revealed in fibrills 20-80 nm thick either with or without cross striation, as well as in microfibrills. Collagen of the III type in the intercellular substance of the arterial wall occurs in nonfibrillar form. Collagen of the IV type is revealed in basal membranes of the smooth muscle cells of the arterial wall, of the muscle fibers and of endothelium of blood capillaries of the skeletal muscle. Collagen of the V type is found as accumulations having various size and form; they localize in many places of the intercellular substance of the arterial wall. A tight contact is revealed between the formations including collagen of the V type with drops of elastin and elastic fibers. A suggestion is made that collagen of the V type participates in formation of elastic fibers.     

Immunological analysis of chick notochord and cartilage matrix development with antisera to cartilage matrix macromolecules

Transverse frozen sections from the postcephalic region of stage 9-16 chick embryos and from the wing bud region of stage 17-31 embryos were stained with antibodies to the major extracellular matrix components of cartilage. These probes included unfractionated A1 and A2 antisera to the major cartilage proteoglycan, affinity-purified purified antibodies to the proteoglycan core protein and to Type II collagen, and a monoclonal antibody to keratan sulfate. In embryos as early as stage 10, notochord stained specifically with the keratan sulfate monoclonal antibody. At this stage the notochord, as well as surrounding tissues, were negative to cartilage proteoglycan and collagen antibodies. Positive staining with the latter probes was coordinately acquired by notochord cells and their accompanying sheath around stage 15, while surrounding tissues remained negative. At this stage, the ventral region of the perispinal cord sheath exhibited light staining with the proteoglycan and keratan sulfate antibodies though failing to react to Type II collagen antibodies. Positive staining of notochord and ventral spinal cord persisted through later developmental stages. As revealed by immunofluorescence, definitive vertebral chondroblasts first emerged at approximately stage 23 and definitive limb chondroblasts at stage 25. The results are discussed in terms of the possible multiple roles of notochord in early embryogenesis.     

Immuno-ultrastructural localization of B-cell-specific monoclonal antibodies B1 and B2

Monoclonal antibodies B1 and B2 are thought to recognize B-lineage restricted antigens, and have been used to define stages of B-cell maturation and characterize B-cell lymphomas. Immunostaining on cryostat sections has revealed a puzzling dendritic or extracellular pattern of staining for B2 within germinal centers and neoplastic follicles. In this study B1 and B2 are localized precisely on hyperplastic and neoplastic lymphoid tissues using immuno-ultrastructural techniques on cryostat sections, cell suspensions, and cell monolayers. B1 and B2 were localized to cell surfaces, including microvillous surface projections, on small and large transformed normal and neoplastic B lymphocytes. B2, in addition to staining in lymphoid cells, was localized to anastomosing cytoplasmic processes of dendritic histiocytes. These findings explain the apparently extracellular localization of B2 in cryostat sections and indicate that patterns of staining for B2 may represent a combination of staining on lymphoid cells and dendritic histiocytes.     

Domain and basement membrane specificity of a monoclonal antibody against chicken type IV collagen

A monoclonal antibody, IV-IA8, generated against chicken type IV collagen has been characterized and shown to bind specifically to a conformational-dependent site within a major, triple helical domain of the type IV molecule. Immunohistochemical localization of the antigenic determinant with IV-IA8 revealed that the basement membranes of a variety of chick tissues were stained but that the basement membrane of the corneal epithelium showed little, if any, staining. Thus, basement membranes may differ in their content of type IV collagen, or in the way in which it is assembled. The specificity of the antibody was determined by inhibition ELISA using purified collagen types I-V and three purified molecular domains of chick type IV collagen ([F1]2F2, F3, and 7S) as inhibitors. Only unfractionated type IV collagen and the (F1)2F2 domain bound the antibody. Antibody binding was destroyed by thermal denaturation of the collagen, the loss occurring at a temperature similar to that at which previous optical rotatory dispersion studies had shown melting of the triple helical structure of (F1)2F2. Such domain-specific monoclonal antibodies should prove to be useful probes in studies involving immunological dissection of the type IV collagen molecule, its assembly within basement membranes, and changes in its distribution during normal development and in disease.     

Immunoelectron microscopic localization of collagens type I, V, VI and of procollagen type III in human periodontal ligament and cementum

We examined the ultrastructural localization of collagens Type I, V, VI and of procollagen Type III in decalcified and prefixed specimens of the periodontal ligament and cementum, by immunoelectron microscopy using ultra-thin cryostat sections. Immunostaining for collagen Type I was pronounced on the major cross-striated fibrils entering cementum and in cementum proper, whereas staining for procollagen Type III was almost exclusively observed on the major fibrils in the periodontal ligament situated more remote from cementum. Reactivity for collagen Type V was limited to aggregated, unbanded filamentous material of about 12 nm diameter that was found mainly in larger spaces between bundles of cross-striated collagen fibrils and occasionally on single microfibrils that apparently originated from the ends of the major collagen fibrils, which may support the concept of this collagen as a component of core fibrils. Collagen Type VI was present as microfilaments appearing to interconnect single cross-striated fibrils. In the densely packed fibril bundles of the periodontal ligament, no collagen type VI was detected. Neither Type V or Type VI collagen was observed in cementum.     

Ezrin immunoreactivity in neuron subpopulations: cellular distribution in relation to cytoskeletal proteins in sensory neurons

Ezrin was first identified as a low-abundance phosphoprotein associated with the cytoskeletal core of microvilli, where it may function as a regulatory protein. We report immunocytochemical evidence for expression of ezrin, or an ezrin-like protein of molecular mass near 80 KD, confined to select populations of neurons, including sensory, motor, and autonomic, during chick embryonic development. We have compared the distribution of anti-ezrin staining with that of other major cytoskeletal proteins in sensory neurons in an effort to identify a possible association of the neural homologue of ezrin with the neuronal cytoskeleton. The diffuse distribution of anti-ezrin staining in the cell soma bore little resemblance to the filamentous staining observed with antibodies to the 68 KD neurofilament protein and alpha-tubulin. F-actin staining with fluorescein-conjugated phalloidin was indistinguishable from the anti-ezrin staining pattern in the soma of cultured neurons, including a peak in staining intensity around the periphery of the cell. Microfilaments in growth cones did not stain with the ezrin antibody. A close correspondence between the anti-ezrin and anti-spectrin staining patterns was found on cryostat sections of dorsal root ganglia, but the anti-spectrin staining was weak and could not be demonstrated in culture. Our findings, primarily from cultured neurons, are not inconsistent with ezrin associating with F-actin, although not with microfilaments found in motile structures such as growth cones.     

Production and characterization of a monoclonal antibody to human type III collagen

Human type III collagen from placenta was isolated and purified for use as an immunogen. A monoclonal antibody was produced which specifically recognizes epitopes unique to type III collagen. The specificity of the antibody was determined by inhibition ELISA, an immunoblot assay, and by immunoprecipitation. Results indicated that the monoclonal antibody recognized only the alpha 1(III) polypeptide chains and did not crossreact with type I, IV, or V collagen. The monoclonal antibody was also used for immunohistochemical localization of type III collagen in tissue sections of human placenta, bovine spleen, and lymph node. In placenta, both large and small blood vessels showed pronounced staining of the tunica media, which contains largely smooth muscle cells, known to synthesize type III collagen. In contrast, the intimal areas and endothelial cells showed no staining with the antibody. In the placental villi, staining was limited to the villous core, where fine fibrillar structures showed strong staining. In lymph nodes, the capsule and pericapsular adipose cells were surrounded by a covering of type III collagen. Within the parenchyma of the node, staining was localized to a branching, reticular array of fine fibers. In the spleen, staining was pronounced in the capsule, splenic trabeculae, and white pulp, where blood vessel staining was especially prominent. The red pulp and splenic sinuses contain little or no type III collagen. The fine network-like or reticular staining pattern found in the lymph node parenchyma is consistent with the staining pattern of the protein reticulin, and suggests that type III collagen may be closely associated with reticulin in certain tissues. Since the role of type III in tissues is unclear, this reagent will be useful in providing new information in this regard.     

Immunofluorescent localization of type-V collagen as a fibrillar component of the interstitial connective tissue of human oral mucosa,artery and liver

Summary Polyclonal antibodies against native human typeV collagen were produced in rabbits and goats. Following purification, crossreaction of the antibodies with highly immunogenic peptides of basement membranes or the interstitial matrix was excluded on the basis of sensitive radioimmunoassays. These antibodies, when applied to cryostat sections of human oral mucosa, liver and arterial walls, never stained basement membranes as did antibodies against type-IV collagen or laminin. On the contrary, we observed delicate arborizing fibers in the interstitial compartment with extensions contacting structures such as subepidermal basement membranes. Arterioles contained a unilamellar sheath of longitudinally oriented fibers limited to the intimal layer. Larger arteries exhibited a multilamellar fibrous fluorescence over the whole intima, whereas the media showed a much weaker staining. The data identified type-V collagen as an interstitial fibrillar collagen rather than a basement membrane collagen, with a tissue pattern completely different from that of collagens types I, III, VI or fibronectin. A reinterpretation of the role of type-V collagen in connective tissue function is warranted.     

Lack of Specificity of Commercially Available Antisera: Better Specifications Needed

The ideal antiserum for immunohistochemical (IHC) applications contains monospecific high-affinity antibodies with little nonspecific adherence to sections. Many commercially available antibodies are “affinity” purified, but it is unknown if they meet “hard” specificity criteria, such as absence of staining in tissues genetically deficient for the antigen or a staining pattern that is identical to that of an antibody raised against a different epitope on the same protein. Reviewers, therefore, often require additional characterization. Although the affinity-purified antibodies used in our study on the distribution of muscarinic receptors produced selective staining patterns on sections, few passed the preabsorption test, and none produced bands of the anticipated size on Western blots. More importantly, none showed a difference in staining pattern on sections or Western blots between wild-type and knockout mice. Because these antibodies were used in most studies published thus far, our findings cast doubts on the validity of the extant body of morphological knowledge of the whole family of muscarinic receptors. We formulate requirements that antibody-specification data sheets should meet and propose that journals for which IHC is a core technique facilitate consumer rating of antibodies. “Certified” antibodies could avoid fruitless and costly validation assays and should become the standard of commercial suppliers. (J Histochem Cytochem 56:1099–1111, 2008)     

Immunogold-silver staining and epipolarized light microscopic detection of phosphoenolpyruvate carboxykinase and glycogen phosphorylase in rat liver

The subcellular distribution of enzymes related to carbohydrate metabolism was determined in sections of paraformaldehyde fixed and polyethylene glycol-1540-embedded rat liver and in cryostat sections. For this purpose, goat anti-rat phosphoenolpyruvate carboxykinase (PEPCK) serum and rabbit anti-rat glycogen phosphorylase (GP) serum were used as primary antibodies to localize the corresponding antigens. The primary antibodies were localized by 5 nm colloidal gold labeled secondary antibodies (either rabbit anti-goat IgG for PEPCK or goat anti-rabbit IgG for GP), and the gold particles were enhanced by silver staining using appropriate development reagents. The silver enhanced gold particles were detected by epipolarized light microscopy. PEPCK and GP immunoreactive molecules were found only in glycogen-containing areas of the cytosome of hepatocytes, and not in other cells. No immunocytochemical staining of hepatocytes was found when normal serum replaced the primary antibody in the procedures. Visio-Bond semithin (0.35–1.0 m) sections provided higher resolution for subcellular immunostaining of PEPCK and GP than cryosections of 10 m. Epipolarized light microscopy provided detection at high sensitivity of the gold-labeled antibody, and combined with transmitted light, allowed simultaneous visualization of the tissue morphology.     

Changes in the synthesis of minor cartilage collagens after growth of chick chondrocytes in 5-bromo-2'-deoxyuridine or to senescence

Analyses were made of the minor collagens synthesized by cultures of chondrocytes derived from 14-day chick embryo sterna. Comparisons were made between control cultures, cultures grown for 9 days in 5-bromo-2'-deoxyuridine (BrdU) and clones of chondrocytes grown to senescence. Separation of minor collagens from interstitial collagens was achieved by differential salt precipitation in the presence of carrier collagens in acid conditions. The precipitate at 0.9 M NaCl 0.5 M acetic acid from control cultures was shown by CNBr peptide analysis to contain only the alpha 1(II) chain of type II collagen, whereas after BrdU treatment or growth to senescence synthesis of only alpha 1(I) and alpha 2(I) chains occurred. The synthesis of type III collagen was not detected. Analysis of the precipitate at 2.0 M NaCl, 0.5 M HAc from control cultures demonstrated the synthesis of 1 alpha, 2 alpha and 3 alpha chains together with the synthesis of short chain (SC) collagen of Mr 43000 after pepsin digestion. After BrdU treatment or growth to senescence alpha chains were isolated which possessed the migration positions on polyacrylamide gel electrophoresis (PAGE), or the elution positions on CM-cellulose chromatography, of the alpha 1(V) and alpha 2(V) chains of type V collagen. In addition, for BrdU-treated but not for control cultures, intracellular immunofluorescent staining was observed with a monoclonal antibody which specifically recognizes an epitope present in the triple helix of type V collagen. Synthesis of short chain (SC) collagen was not detected after BrdU treatment or growth to senescence. These results suggest that chick chondrocytes grown in conditions known to cause switching of collagen synthesis from type II to type I collagen also undergo a switch from the synthesis of 1 alpha, 2 alpha and 3 alpha chains to the synthesis of the alpha 1(V) and alpha 2(V) chains of type V collagen. It appears that there are several cartilage-specific collagens which together undergo a regulatory control to the synthesis of collagens typical of other connective tissues.     

Monoclonal antibodies to luteinizing hormone-releasing hormone: production, characterization, and immunocytochemical application

Luteinizing hormone-releasing hormone (LHRH) was conjugated to bovine thyroglobulin and used to immunize a BALB/c mouse. Spleen lymphocytes were subsequently fused to SP2/0 myeloma cells and two of the resulting hybridoma clones were found to produce high titer antibodies to LHRH (HU4H and HU11B); both belonged to the IgG1 subclass. Characterization of the monoclonal antibodies revealed that HU4H and HU11B have conformational and sequential specificity to LHRH, respectively, and that neither one shows significant immunoactivity with pro-LHRH. The value of these antibodies in immunocytochemical applications is demonstrated by their ability to cause intense specific staining of LHRH neuronal cell bodies and fibers in brain sections from several mammalian species.