Axioplan 2 epi fluorescence upright microscope

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.

After fixation with 4% PFA for 18 min, cells were rinsed three times in MT-PBS. Cells were blocked with 10% normal goat serum with 0.3% Triton X-100 in MT-PBS for 60 min at RT, followed an incubation with primary antibodies against GFAP (1:200, mouse, catalog #MAB360, Millipore; 1:200, rabbit, catalog #Z03374, DAKO), MBP (1:50, mouse, catalog #MAB381, Millipore; 1:100, rat, catalog #ab980, Millipore), or rabbit anti-Neurofilament (1:200; catalog #AB1987, Millipore). Cells were then rinsed with MT-PBS followed by the appropriate fluorophore-conjugated secondary antibodies (all 1:500 in antibody diluent, Thermo Fisher Scientific) for 60 min at RT. Cells were rinsed twice in MT-PBS before adding Hoechst (1:10,000 in MT-PBS; catalog #33342, Invitrogen) for 10 min. Cells were rinsed twice in MT-PBS and mounted with Cytomation fluorescence mounting medium (Dako) on SuperFrost Plus glass slides (Thermo Fisher Scientific). Six fields per culture, and three technical replicate from a minimum of three animals per condition or genotype were analyzed, and images captured by a Carl Zeiss Axioplan 2 epifluorescence upright microscope.