Berit Hølund

The influence of fixation and tissue preparation on the immunohistochemical demonstration of fibronectin in human tissue

SummaryThe influence of fixation and tissue preparation on the immunohistochemical localization of human fibronectin in gastrointestinal tract tissue has been examined using indirect immunoperoxidase technique. The most optimal staining result with strong intensity and well defined localization was obtained on frozen sections of unfixed material. Nearly identical results with improved morphology were obtained when staining paraffin sections of tissue fixed in 96% ethanol, 96%+1% acetic acid and absolute acetone. All other fixatives tested, 10% neutral buffered formalin, Lillies AAF, Bouins fixative, Clarkes fixative, 4% formaldehyde, 4% formaldehyde+ 0.5% cetylpyridiniumchloride (F-CPC), 4% formaldehyde +0.1% glutaraldehyde gave unsatisfactory results. However, proteolytic digestion with pepsin of paraffin sections prior to staining of buffered formalin and F-CPCfixed material gave results comparable with those obtained on unfixed frozen sections as regards definition of the staining whereas staining intensity was decreased in some degree. No improvement was observed when using proteolytic digestion of tissue fixed in other fixatives.

Demonstration of fibronectin in normal and injured aorta by an indirect immunoperoxidase technique.

SummaryThe presence and localization of fibronectin in normal and mechanically injured aorta in rabbits was studied using an indirect immunoperoxidase technique on tissue specimens fixed in formaldehyde, embedded in paraffin and pretreated with pepsin. The effect on staining quality of treatment with testicular hyaluronidase prior to immunoperoxidase staining was also examined. In the intima from normal aorta fibronectin was present in the subendothelial basal layer, along the internal and external elastic laminae, around and between the smooth muscle cells of the media and along the collagen and elastic fibres in the adventitia. Sixteen days after a single mechanical dilatation of the descending thoracic aorta all animals developed gross atherosclerotic-like changes. Microscopic examination revealed prominent neo-intimal hyperplasia with subendothelial, cushion-like thickenings but no medial or adventitial alterations. Fibronectin, in increased amounts, was found between and around the endothelial cells and in the subendothelial thickenings between the proliferating smooth muscle cells in relation to the fine, thin elastic and argyrophilic fibres. In the media and adventitia the amount and distribution of fibronectin was indistinguishable from uninjured control aortas. Treatment with testicular hyaluronidase before immunoperoxidase staining resulted in a higher staining resolution in normal and injured aorta. The conspicuous observation in the present study is that fibronectin exclusively accumulates in areas of tissue repair. The origins and functions of fibronectin during tissue injury and repair are discussed.

Demonstration of fibronectin in human articular cartilage by an indirect immunoperoxidase technique.

SummaryFresh frozen tissue sections of human articular cartilage was treated without and with human testicular hyaluronidase (2×106 units/l) for 60 min at 37° C and stained by the indirect immunoperoxidase technique with rabbit antihuman fibronectin. The rabbit antihuman fibronectin was purified by affinity chromatography on human fibronectin-Sepharose. Fibronectin was only found on the acellular surface of the articular cartilage in tissue sections not treated with hyaluronidase. In this surface layer, probably identical to “lamina splendens”, the arrangement of fibronectin was as a membrane. No collagen was seen in this area by van Gieson staining. No staining for fibronectin was found in the cartilage matrix or in the chondrocytes. Treatment of the cartilage tissue with hyaluronidase resulted in visualization of high amount of fibronectin in the cartilage matrix, with the highest intensity around the chondrocytes. The staining of the acellular surface layer of the articular cartilage was identical with the results obtained without hyaluronidase treatment. These results indicate that articular cartilage is rich in fibronectin probably in complex with hyaluronic acid, and that the chondrocytes produce fibronectin in situ. It also demonstrates the steric hindrance of hyaluronic acid aggregates in diffusion of the antibody and the value of hyaluronidase treatment of tissue before demonstration of fibronectin.

The value of hyaluronidase treatment of different tissues before demonstration of fibronectin by the indirect immunoperoxidase technique

SummaryThe influence of testicular hyaluronidase treatment on the immunohistochemical localization of fibronectin in different tissues (human articular cartilage, large intestine, synovial membrane and experimental granulation tissue) as well on frozen as on formaldehyde fixed, paraffin embedded tissue, has been studied using the indirect immunoperoxidase technique. Pretreatment with hyaluronidase is essential in demonstrating fibronectin in frozen sections of human articular cartilage. In the other tissues examined treatment with hyaluronidase was not essential, but gave a more optimal staining quality. The effect of hyaluronidase treatment was to some extent dependent on the duration of treatment. In formaldehyde fixed, paraffin processed tissue the improvement with hyaluronidase treatment was only seen when the hyaluronidase followed pepsin digestion of the deparaffinized tissue sections.

Immunohistochemical study of possible changes in keratin expression during neoplastic transformation of the uterine mucosa

The present study aimed to examine possible changes in keratin expression during neoplastic transformation of the uterine mucosa and possible differences in keratin expression between endocervical and endometrial adenocarcinomas. Routinely processed specimens with normal morphology or neoplastic changes were stained immunohistochemically using 5 commercial antibodies to keratinfilaments of molecular weight 39–58 kD: CAM 5.2, RCK 102, MCA 144, PKE and PRE. We generally found a change in keratin expression during the neoplastic transformation, consisting of pronounced heterogeneity compared with normal epithelia. In distinguishing koilocytic atypia from CIN, RCK 102 (52.5, 58 Kd) may prove helpful as it stains neoplastic cells strongly and shows no reaction in koilocytes. Staining with the antibody CAM 5.2 (reactive with 39, 43, 50 kD filaments) may aid in distinguishing between cervical and endometrial adenocarcinomas. The former is stained uniformly; the latter shows a more variable staining.

Sequential appearance of fibronectin and collagen fibres in experimental arthritis in rabbits

SummaryThe sequential changes in the presence of fibronectin in the synovial membrane during the development of antigen-induced arthritis in rabbits were studied using an indirect immunoperoxidase technique on the tissue specimens fixed in formaldehyde, embedded in paraffin and pretreated with pepsin and testicular hyaluronidase. The relation to the distribution of fibronectin and connective tissue fibres, demonstrated as either argyrophilic or red by van Gieson method, was studied. Initial after the induction of the arthritis the synoviocytes became increased in size and number. The subsynoviocytial tissue was invaded by granulocytes and the number of vessels was increased. Fibronectin in increased amount was seen around the lining cells. After 2–4 weeks a markedly reduced amount of granulocytes were seen together with an increase in the number of macrophages. At this stage, fibronectin was also found together with argyrophilic fibres in the subsynoviocytial connective tissue. After 8–13 weeks the synovial membrane was found hypertrophic and folded. The lining layer was unchanged, but in the subsynoviocytial tissue lymphocytes and plasma cells were more focally arranged. At that time fine fibres, stained by the van Gieson method, were present together with fibronectin and argyrophilic fibres in the subsynoviocytial tissue.The morphological change and the distribution of fibronectin in experimentally induced arthritis correlates temporally to the morphological change and the presence of fibronectin found in experimentally induced granulation tissue.

Differentiation between metaplastic carcinomas and sarcomas of the human female breast by fibronectin

The distribution pattern of fibronectin in metaplastic carcinomas, stromal sarcomas, malignant cystosarcoma phyllodes tumours and histiocytic type lymphomas of the human female breast has been studied using the indirect immunoperoxidase technique on formalin fixed paraffin embedded tissue. Fibronectin was demonstrated as intensely stained strands between tumour cells forming an irregular network in metaplastic carcinomas and lymphomas. Stromal sarcomas and the malignant stromal component of the phyllodes tumours exhibited, in contrast, a uniform staining throughout tumour cells and stroma which was weaker than in adjacent normal-looking connective tissue. We suggest that the intense staining reaction of metaplastic carcinomas is due to the scirrhous reaction generally associated with invasive human breast carcinomas. The advantage of using fibronectin as a diagnostic tool in the differentiation of carcinoma/lymphoma versus sarcoma is the fact that the antigen is a stromal marker and its staining intensity is not influenced by the morphology or degree of differentiation of non-mesenchymal tumours.

In vivo demonstration of cytoplasmic fibronectin in human breast carcinomas

The distribution pattern of fibronectin in 24 invasive human breast carcinomas has been studied using the indirect immunoperoxidase technique. A positive cytoplasmic staining reaction was observed in 16 tumours. Well-differentiated carcinomas showed weak or no staining, whereas all moderately or poorly differentiated carcinomas and one signet ring cell carcinoma contained fibronectin positive tumour cells with moderate or strong staining. The staining intensity was positively correlated to degree of anaplasia with the exception of two moderately differentiated duct carcinomas and the two medullary carcinomas, which were only slightly positive. Non-attached independently growing tumour cells stained more intensely than tumour cells in clusters. Pericellular fibronectin was found in only one carcinoma of medullary type. In normal ducts and glands it was seen at the stromal-epithelial junction corresponding to the basement membrane, around myoepithelial cells and along the luminal border. The results support the findings of several in vitro investigations that breast tumour cells synthesize fibronectin. It also suggests that cytoplasmic fibronectin expression might be an indicator of tissue differentiation in non-solidly growing invasive duct carcinomas of the human mammary gland.