Ab182422

Immunohistochemical (IHC) staining was conducted following standard procedures, which included fixation, deparaffinisation, hydration, antigen retrieval, blocking and incubation with primary antibodies (4°C overnight) and biotinylated secondary antibodies. The experiment utilised the following primary antibodies: anti‐CD68 (Abcam, ab283654), anti‐ABCA1 (Abcam, ab18180) and anti‐CD163 (Abcam, ab182422). IHC staining was evaluated as previously reported.
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The pancreatic cancer tissue microarray above was used to evaluate the expression of CD59 and CD163 in tumor and peritumor tissue. The sections were then deparaffinized in xylene, rehydrated according to standard histopathological procedures and stained with hematoxylin and eosin. The slides were then incubated with 1:200 dilutions of CD59 antibody (HPA026494, Sigma-Aldrich) and CD163 antibody (ab182422, Abcam). The stained tissues were scored by two pathologists blinded to the patient information. The expression level of CD59 was evaluated by the H-score^{14 (link)}, a widely used evaluation criterion for immunostaining that involves multiplying a proportion score and an intensity score^{15 (link)–17 (link)}. The H-score value with the largest Youden’s Index for each variable within the receiver operating characteristic (ROC) curve was selected as the cut-off. The specimens with an H-score above or equal to the cut-off value were defined as those with high expression of CD59, whereas the others were regarded as having low CD59 expression. CD163 is a commonly accepted marker for tumor-promoting TAMs^{11 (link)}. The CD163-positive macrophages in the stained sections were counted using ImageScope software, and the density was estimated (per square millimeter) at a higher magnification (×200). The cut-off value was selected by the ROC curve as mentioned above.

Three ventricular sections from each rat were incubated with individual primary antibodies to CD68 (GB11067; 1/3,000; Servicebio Technology, Wuhan, China), CD163 (ab182422; 1/500; Abcam), or CD86 (13395‐1‐AP; 1/500; Wuhan Sanying). Subsequently, sections were incubated with secondary antibodies conjugated with fluorescence. The slides were washed three times with PBS, incubated in 4′,6‐diamidino‐2‐phenylindole (G1012; 1:1; Servicebio Technology) for 10 min, and then dried and coverslipped for evaluation. Fluorescent signals of 5 random nonoverlapping fields were captured with a fluorescence microscope (Nikon Eclipse C1; Nikon, Tokyo, Japan). All images were analyzed by Image‐Pro Plus 6.0 software. Quantification was performed by calculating the percentage of the positively stained area to the total area at a ×400 magnification.

For histologic evaluation, the fixed right lung tissues were embedded in paraffin, sectioned (4 μm) onto glass slides and stained with H&E for structured observation and Masson’s trichrome for detection of collagen deposits. The development of pulmonary lesions was scored by two pathologists blind to the study design. For immunohistochemistry staining, the sections were permeabilized in 1× PBS containing Triton X-100 (0.1%) for 10 minutes and then probed with antibodies against CD206 (ab64693, Abcam, Cambridge, Massachusetts, USA), CD163 (ab182422, Abcam), CD86 (BS9900M, Bioworld, St. Louis Park, Minnesota, USA), iNOS (ab178945, Abcam), CD68 (ab213363, Abcam), TGF-β1 (ab215715, Abcam) and αSMA (ab32575). For the immunofluorescence assay, slides were first labeled with a mouse-derived antibody against CD206 (ab8918, Abcam) and a rabbit-originated GRP78 (ab21685, Abcam) antibody followed by staining with an Alexa Fluor 595-labelled anti-mouse and Alexa Fluor 488-conjugated anti-rabbit antibodies (Thermo Fisher Scientific, Inc., Waltham, Massachusetts, USA). Images were captured using a Zeiss Axio upright fluorescent microscope.