title: How to obtain good morphology and antigen detection in the same tissue section?
abstract: Most human and animal biopsy samples are routinely embedded in paraffin since this enables the pathologist or researcher to obtain excellent morphology and simplifies storage. Nevertheless, in many cases, the antigen of interest cannot be detected in paraffin section. The alternative available for good immunohistochemistry is preparation of cryosections, which usually provide decent antigen preservation and are frequently used for immunofluorescence. However, cryosections often do not provide efficient morphological details of tissues and cells for pathologic evaluation. In order to obtain good antigen preservation and improve tissue and cell morphology after freezing, we tested three different fixations and freezing methodologies and compared them to routine formaldehyde fixation and paraffin embedding. As a model system, we selected the epithelium of the rat urinary bladder and trachea. On all samples, haematoxylin and eosin staining was performed as well as immunofluorescence with antibodies against tight junction protein ZO-1 and against intermediate filament cytokeratin 7. The best compromise between morphology and immunofluorescence was obtained with "sucrose impregnation prior to freezing" method. Moreover, this procedure is also quicker in comparison to standard paraffin section preparation. To check the clinical relevance of our study, this method was used for human biopsy samples of neoplastic urothelial and bronchial mucosa lesions. Besides good immunofluorescence results, the morphology of these samples was well preserved. We therefore propose that cryosection preparation with sucrose impregnation prior to freezing should be further exploited in other clinical and veterinary applications, since it enables good morphology and antigen preservation.
authors.affiliation: Institute of Cell Biology, Faculty of Medicine, University of Ljubljana, Vrazov trg 2, 1000, Ljubljana, Slovenia. dasa.zupancic@mf.uni-lj.si.
authors.forename: Daša
authors.initials: D
authors.lastname: Zupančič
authors.affiliation: Department of Pulmonary and Allergic Diseases, University Medical Centre Ljubljana, Zaloška cesta 2, 1000, Ljubljana, Slovenia.
authors.forename: Marjeta
authors.initials: M
authors.lastname: Terčelj
authors.affiliation: Department of Urology, University Medical Centre Ljubljana, Zaloška cesta 2, 1000, Ljubljana, Slovenia.
authors.forename: Bojan
authors.initials: B
authors.lastname: Štrus
authors.affiliation: Institute of Cell Biology, Faculty of Medicine, University of Ljubljana, Vrazov trg 2, 1000, Ljubljana, Slovenia.
authors.forename: Peter
authors.initials: P
authors.lastname: Veranič
mesh_terms: D000818:Animals; D016616:Cryoultramicrotomy / Q000379:methods*; D005455:Fluorescent Antibody Technique; D005557:Formaldehyde; D006801:Humans; D007150:Immunohistochemistry; D016612:Paraffin Embedding / Q000379:methods; D016707:Tissue Fixation / Q000379:methods
chemical_list: D005557:Formaldehyde
keywords: Cryosections; Immunofluorescence; Morphology; Paraffin sections; Sucrose impregnation