Phosphotyr641stat6

Sections were deparaffinized and rehydrated prior to antigen retrieval as described (23 (link)). Tissue sections were then incubated overnight at 4°C with 1:50 dilution of anti-arginase I (ArgI, Santa Cruz Biotechnology) primary antibody followed by 1:1000 dilution of Alexa 568-conjugated anti-goat secondary antibody. A mixture of anti-NOS2 (BD Transduction Labs; 1:50 dilution) and anti-F4/80 (ABD-Serotec; 1:50 dilution) primary antibodies were then applied for 1 h at 37°C, followed by 20 min incubations with Alexa 488-conjugated anti-rabbit and Alexa 680-conjugated anti-rat secondaries. Nuclei were stained with DAPI-containing mounting media (Vector Laboratories). Images were obtained with a digital deconvolution microscopy imaging system attached to a Zeiss Axioplan 2 epi-Fluorescence upright microscope. Macrophages were identified by positive F4/80 staining and morphology. Total pixel counts/macrophage were calculated for ArgI, NOS2, and F4/80 immunofluorescence (~50/animal) using ImageJ software (38 (link)), and ArgI and NOS2 values were normalized to F4/80 staining. To confirm ArgI⁺ M2 programing, adjacent sections were subjected to a similar IF protocol substituting an antibody against M2 marker phosphoTyr⁶⁴¹STAT6 (Cell Signaling, 1:50 dilution) for NOS2. Fluorescence intensity was calculated similarly and phosphoSTAT6/ArgI ratios determined.