Anti human ki 67 clone mib 1

Immunohistochemistry using sections of paraffin-imbedded tumors was carried out as previously described [30] (link). Briefly, after antigen retrieval (microwave, citrate buffer, ph = 6), mouse anti-YKL-40 or corresponding isotype control (see above) was used as first antibody, respectively, followed by incubation with biotinylated rabbit anti-mouse (Dako, Glostrup, Denmark) and visualization using the streptavidin-alkaline-phosphatase based Vectastain ABC kit (Vector, Burlingame, CA, USA). Slides were scanned by a Mirax microscope (Zeiss, Jena Germany) and the Mirax Viewer (Zeiss) software was used to take images. Other antibodies used for immunohistochemistry (with corresponding biotinylated secondary antibodies (all Dako)) were: Anti murine EDG-1 (S1P1) (Santa Cruz, Heidelberg, Germany), anti-murine CD45 (clone 30-F11, BD) and anti-human Ki-67 (clone MIB-1, Dako). Images of four randomly chosen fields of vision (not containing necrotic areas) were taken with the viewer software. Human Ki-67 or murine CD45 positive cells were counted and murine S1P1 positive areas quantified using the ImageJ software (Wayne Rasband, NIH, USA). Positive cells or percent positive area per field of vision were calculated for each tumor.
Histochemical Masson-Goldner staining of paraffin-imbedded tumors was performed as described [33] .

Our immunohistochemical study, performed on recurrent OOC samples included the analysis of two well-known markers (p53 and Ki67), both of which are implicated in tumorigenesis and cell proliferation [6 (link)]. Briefly, 5-μm sections were cut from the original paraffin-embedded blocks and mounted on poly-L-lysine-coated glass slides prior to immunohistochemical staining, performed using monoclonal antibodies: mouse antihuman p53 (clone DO-7, dilution 1:50, Dako®, Glostrup, Denmark) and antihuman Ki67 (clone MIB-1, dilution 1:100, Dako®, Glostrup, Denmark). Immunostaining was visualized using the high-pH EnVision FLEX system (Dako®, Glostrup, Denmark); hematoxylin was used for counterstaining and for both techniques tonsil sections with oropharyngeal epithelium were employed as positive staining controls and the negative controls were mock-stained test sections (the primary antibody was replaced with PBS).