Ncl cd163

Immunostaining was performed on 4 μm thick formalin fixed paraffin embedded tumor sections using anti-MGL antibodies synthesized in our laboratory (IG6.6, IgG2a) (23 (link)) and anti-CD163 antibody (NCL-CD163 LeicaBiosystems, IgG1). Antigen retrieval was performed using Tris–ethylenediaminetetraacetic acid (EDTA) buffer (10 mM Tris plus 1 mM EDTA pH 9.0). Antigen retrieval was followed by overnight incubation at room temperature with anti-MGL and anti-CD163 antibody diluted in 1% w/v bovine serum albumin in PBS. Alexa Fluor labeled Goat-anti-mouseIgG1-A488 (CD163) and Goat-anti-mouseIgG2a-A546 (both from Invitrogen, Life Technologies, Carlsbad, USA) were incubated at room temperature for 1 hour. Slides were mounted using VectaShield mounting medium containing DAPI. Images were obtained using an LSM700 confocal laser scanning microscope containing an LCI Plan-Neofluar 25 × /0.8 Imm Korr DIC M27 objective (Zeiss, Göttingen, Germany).

FFPE tissue sections (10 μm) were de-paraffinized and rehydrated through a graded alcohol series. Antigen retrieval was performed on FFPE slides using 1X Target Retrieval Solution (Dako, Carpinteria, CA). Endogenous peroxidase was blocked using 3% hydrogen peroxide for 10 minutes. The slides were incubated overnight with primary antibodies against Fascin (PA0420, Leica Biosystems, Buffalo Grove, IL, as it is), CD207 (NCL-LANGERIN-U, Leica Biosystems, Buffalo Grove, IL, 1:100 dilution), CD1A (PA0553, Leica Biosystems, Buffalo Grove, IL, as it is), Factor XIII (PA0449, Leica Biosystems, Buffalo Grove, IL, as it is), CD163 (NCL-CD163, Leica Biosystems, Buffalo Grove, IL 1:1500 dilution), and CD-68PGM1 (M0876, Agilent Technologies, Santa Clara, CA 1:300 dilution) at 4°C. Following incubation with secondary antibodies at room temperature for 1 hour, immunoreactivity were detected using diaminobenzidine (Innovex Biosciences, Richmond, CA) as a chromogen. Slides were counter-stained with hematoxylin before imaged with Olympus BX51 microscope fitted with a DP26 camera.