Mouse anti cd68

Formalin-fixed, paraffin-embedded lung tissue sections (left lung lobe, 5 μm) were deparaffinized, antigen-unmasked by heating tissue sections in universal antigen retrieval reagent (Cell Signaling Technology, Danvers, MA), and used to perform immunofluorescent staining. The primary antibodies used were rabbit anti-F4/80 (1:100, Thermo Fisher Scientific) or mouse anti-F4/80 (1:100, Santa Cruz Biotechnology, Santa Cruz, CA), mouse anti-CD68 (1:100, Novus Biologicals, Centennials, CA), mouse anti-CD206 (1:100, Abcam, Cambridge, MA, USA), rabbit anti-ALOX5AP (1:100, Abcam), and mouse anti-ALOX15 (1:200, Abcam) antibodies. Images were taken in three to five randomly selected fields per lung slice, three lung slices per mouse, using a Zeiss LSM 780 confocal microscope with a 63× magnification lens (Carl Zeiss Microscopy, Jena, Germany). More than 500 cells from captured microscopic images per each treatment were counted using the ImageJ software (NIH, Bethesda, USA) and the number of cells with double positive staining per every 100 cells were presented as mean ± SEM (n = 3).

Paraffin-embedded aorta sections mounted slides were subjected to heat-induced antigen epitope retrieval with citrate buffer (Thermo Scientific, MA). The slides were incubated overnight at 4°C with primary antibodies for anti—MMP-9 (Rabbit anti-Mouse) (Invitrogen, Carlsbad, CA), Rabbit anti-Mouse anti—MMP-2 (R & D Systems, Minneapolis, MN), Mouse anti- CD-68 (Novus, Cambridge, United Kingdom), anti-Mouse Mac-2 (Cedarlane, Burlington, Canada), and anti-mouse TGFβ1 (R & D Systems, Minneapolis, MN). The sections were incubated with relevant secondary antibodies (Goat anti- Mouse) (Invitrogen, Carlsbad, CA). IHC staining was completed with IHC kit (Enzo Life Sciences, NY). Slides were visualized by 3-Amino-9-ethylcarbazole (AEC) (Vector Laboratories, Burlingame, CA) chromogens followed by an appropriate counterstain.