Fig 1: PD-L1-positive M2 macrophages surrounding PD-L1-negative tumors. Microscope magnification: (A, C, E, G, I: 200-fold; B, D, F, H, J: 400-fold). Anti-PD-L1 antibody (28-8) (Dako SK005), anti-CD68 antibody (Abcam Cat# ab955), anti-CD206 antibody (Abcam Cat# ab64693) and anti-CD-8 antibody (Roche Cat# 7904460) were used for immunostaining. A, B: Hematoxylin and Eosin staining. The blue arrowhead indicates lung cancer cells. The yellow arrow indicates macrophages. C, D: The tumor did not express PD-L1. Instead, the surrounding cells showed high PD-L1 expression. E, F: The PD-L1-positive cells were CD68-positive macrophages. G, H: CD206 staining was also positive in the macrophages. I, J: CD8-positive lymphocytes were observed in the vicinity of the cancer cells.
Fig 2: CD68+ (a, b) and CD163+ (c, d) macrophages in the sections of axillary lymph nodes (ALNs), using IHC staining, at 400x magnification. Briefly, heat-mediated antigen retrieval was performed using citrate buffer, pH 6 (20 mins). The sections were then incubated with MAbs to CD68 (Abcam, ab955) at a 1 : 300 dilution for 30 mins at RT, MAbs to CD163 (Abcam, ab74604) at a prediluted concentration for 30 mins at RT. Polymeric HRP-linker antibody conjugate was used as secondary antibody. DAB chromogen was used to visualize the staining. The sections were counterstained with haematoxylin. (a, c) Low percentage of CD68+ and CD163+ macrophages; (b, d) high percentage of CD68+ and CD163+macrophages. The positive brown membrane-stained cells in tumour-free medullary areas of ALNs were quantified as the average % of all cells (5 HPFs).
Fig 3: CD68+ (a, b) and CD163+ (c, d) macrophages in the sections of LLABCs, using IHC staining, at 200x magnification. Briefly, heat-mediated antigen retrieval was performed using citrate buffer, pH 6 (20 mins). The sections were then incubated with MAbs to CD68 (Abcam, ab955) at a 1 : 300 dilution for 30 mins at RT, MAbs to CD163 (Abcam, ab74604) at a prediluted concentration for 30 mins at RT. Polymeric HRP-linker antibody conjugate was used as secondary antibody. DAB chromogen was used to visualize the staining. The sections were counterstained with haematoxylin. (a, c) Low level of CD68+ and CD163+ macrophage infiltration; (b, d) high level of CD68+ and CD163+macrophage infiltration. Tumours were classified as low level of infiltration when the positively brown membrane-stained cells were scattered or continuous along the tumour margin but did not extend from the tumour front (TF) for more than one cell layer. Extension for two or more layers from the TF was classified as a high level of infiltration.
Fig 4: M2 macrophages surrounding the lung tumors. Double staining with anti-CD68 antibody (Abcam Cat# ab955) and anti-CD206 antibody (Abcam Cat# ab64693) was performed. The anti-CD68 antibody appeared red due to the secondary fluorescent conjugated antibody (Alexa Fluor® 594 Abcam ab150116). The anti-206 antibody appeared green due to the secondary fluorescent conjugated antibody (Alexa Fluor® 488 Abcam ab150077). Images were overlaid to demonstrate co-localization. Blue: DAPI, Red: CD68, Green: CD206. The white dotted line indicates tumor cells.
Supplier Page from Abcam for Anti-CD68 antibody [KP1]