| Study protocol for testing the immunoreactivity of anti-CD44 antibodies on paraffin sections using wet autoclave and microwave pretreatment for antigen retrieval. Patients material: Routinely formalin fixed and paraffin embedded biopsy material of 3 squamous cell carcinomas of the oral cavity, 3 breast carcinomas and colonic adenocarcinomas retrieved from the files of the Institute of Pathology, University of Münster Handling of the sections: 1-2 µm thick paraffin sections were cut and mounted on poly-L-lysine coated glass slides. In cases of breast cancer different "glue" techniques were tested because of the frequent loss of sections during pretreatment procedures: the best results were achieved using either concentrated poly-L-lysine coated slides (without dilution layered over the precleaned glass slides) or by using Super Frost or Super Frost Plus slides (Menzel, Germany) Dewaxing of the sections (as for routine immunohistochemistry):
2x15 minutes in xylene, 3 minutes in ethanol abs, 3 minutes in 90% ethanol, 3 minutes in 70% ethanol, 5 minutes in distilled water Antigen retrieval using the wet autoclave protocol (Bankfalvi et al. J. Pathol. 174, 223 (1994)). Dewaxed sections were immersed in sodium citrate buffer (0.01 M Na-citrate monohydrate, pH 6.0) in plastic Coplin jars and incubated in a Goessner Laborautoklav GLA 40-2 for 5 minutes. |
