Anti cd68 pg m1

Sections (3‐μm thick) obtained from paraffin‐embedded tumor samples were immunohistochemically stained. The following mouse monoclonal antibodies were used: anti‐CD204 (SRA‐E5; TransGenic), anti‐CD163 (10D6; Novocastra, Newcastle, UK), anti‐CD68 (PG‐M1; DAKO, Glostrup, Denmark) and anti‐Ki‐67(MIB‐1; DAKO, Glostrup, Denmark).20 After the samples were reacted with these first antibodies, the samples were incubated with HRP‐labeled secondary anti‐mouse antibody (Nichirei, Tokyo, Japan). The reaction was visualized using the Diaminobenzidine (DAB) system (Nichirei). Normal mouse immunoglobulin (DAKO) was used as a negative control and no signal was observed in these sections. CD68‐, CD163‐ and CD204‐positive cells were counted in four randomly selected areas of a high power field (×200) of a microscope by two pathologists (S.T. and M.Y.) who were blinded to information about the patients’ backgrounds or their prognosis. The data of the Ki‐67 labeling index was previously counted by our research group.21, 22 Double immunostaining of CD204, CD68, CD163 and Ki‐67 was performed as previously described.14 In brief, the sections were reacted with anti‐Ki‐67, CD163 or CD204 antibodies and visualized with DAB. After the sections were washed with citrate buffer (pH 2.2), they were reacted with anti‐CD68 or CD204 antibodies and visualized with HistoGreen (Linaris, Heiderberg, Germany).