Anti cd68 kp1

THP-1 monocytes were seeded at 100 000 cells/well in 24-well plates containing a coverslip and were differentiated as described here above. Undifferentiated monocytes were attached on coverslips by drying a PBS drop containing 100 000 cells. For labeling, cells were fixed for 10 min with paraformaldehyde 4 % in cold PBS, washed three times with 2 % PBS–BSA (bovine serum albumin) and incubated overnight at 4 °C with the primary antibody 1:100 diluted in 2 % PBS-BSA: anti-CD68 (KP1) from Abcam (ab955), anti-CD71 (H300) from Santa Cruz (sc-9099), anti-CD36 (H300) from Santa Cruz (sc-9154), anti-CD14 (1H5D8) from Abcam (ab181470). Cells were washed three times with 2 % PBS–BSA and then incubated for 1h with the secondary antibody. Alexa Fluor-488-conjugated anti-rabbit IgG antibody (Molecular Probes, #A11034) was used at 1/1000 dilution. Cells were then washed three times with PBS, the coverslips were mounted in Mowiol (Sigma) and observed with a confocal microscope (SP5, Leica).

TILs were assessed using H and E slides in FFPE sections obtained through core needle biopsies prior to therapy and through surgical specimens after treatment at surgery. Separate individual slides were stained with anti-CD56 (123C3.D5 + 123A8, Abcam plc, Cambridge, MA), anti-CD3 (SP7, Abcam plc), anti-CD8 (SP16, Abcam plc), anti-FoxP3 (1054C, R and D systems), and anti-CD68 (KP1, Abcam plc) antibodies. All slides were prepared and stained by the UAMS pathology core. The percentage of TILs was determined according to the method proposed by the International Immuno-Oncology Working Group [31 (link), 32 (link)]. The same strategy was used to score various lymphocytes including CD56- and CD3-positive TILs. Slides were independently evaluated by two pathologists (SPO and GRP). Images were analyzed using a Nikon Eclipse Ni microscope at 20X magnification. Photomicrographs were taken with a Nikon DS-Fi3 microscope camera using the NIS-Elements D software from Nikon.