Peroxidase dab bond polymer refine detection system

After sectioning of 4 µm slides from the formalin-fixed, paraffin-embedded blocks, deparaffinization in xylene and rehydration in a series of decreasing concentration of ethanol were done. Antigen retrieval using either the Bond Epitope Retrieval Solution 1 (pH 6) or the Bond Epitope Retrieval Solution 2 (pH 9) (Leica Microsystems, Wetzlar, Germany) at 99-100°C for 20–30 min was performed. The slides were treated with S-100 (1 : 300, clone S-100, DAKO, Carpinteria, California, USA, incubation time: 30 min in room temperature), vimentin (prediluted, clone V91, DAKO, incubation time: 30 min in room temperature), CD31 (1 : 30, clone JC70A, DAKO, incubation time: 30 min in room temperature), MDM2 (1 : 200, clone IB10, Novocastra, Wetzlar, Germany, incubation time: 45 min in room temperature), and p16 (1 : 10, clone G175-405, DAKO, incubation time: 30 min in room temperature), separately. Immunostaining was performed on Leica BOND-MAX autostainer (Leica Microsystems) and we used peroxidase/DAB Bond Polymer Refine Detection System (Leica Microsystems) for visualization. Cytoplasmic staining for vimentin and CD31 and nuclear staining for MDM2 are regarded as positive stain whereas, for S-100 and p16, both nuclear and cytoplasmic stains are required.

Immunohistochemistry was performed on serial 4-μm thick paraffin sections. The paraffin sections were deparaffinized in xylene and rehydrated in a descending ethanol series. Bond Epitope Retrieval Solution 1 [pH ~ 6] or Bond Epitope Retrieval Solution 2 [pH ~ 9] (Leica Microsystems, Wetzlar, Germany) were used for antigen retrieval. Mouse monoclonal WT1 antibody (dilution 1:100, Clone 6 F-H2, Dako) was applied on the slides. Immunohistochemical staining was performed with a Leica Bond-MAX™ autostainer (Leica Microsystems, Berlin, Germany) and the peroxidase/DAB Bond™ Polymer Refine Detection System (Leica Microsystems) was used for visualization.