Rat anti mouse neutrophil specific antibodies

Paraffin embedded lung sections were cut at 5 μm, de-paraffinized, rehydrated, and stained as previously described [33 (link)]. Sections were labeled with primary rat anti-mouse F4/80 antibodies (Bio-Rad, Portland, ME, USA, 1:20 dilution), and rat anti-mouse neutrophil-specific antibodies (Abcam, Cambridge, MA, USA,1:200 dilution) following secondary biotinylated goat anti-rat antibodies (Dako North America Inc, Santa Clara, CA, USA). The heat-induced epitope retrieval method was used for both antibodies. The sections were incubated with streptavidin-peroxidase and developed using an 3-amino-9-ethylcarbozole (AEC) kit (both from Thermo Fisher Scientific, Waltham, MA, USA). Sections were counterstained with hematoxylin (Vector Laboratories, Inc., Burlingame, CA, USA). For the controls, the primary antibody was replaced with equivalent concentrations of the rat non-specific serum. Images were acquired by Olympus IX51 microscope with Olympus DP72 camera (both from Olympus Corporation, Waltham, MA, USA). Quantification of lung infiltrating macrophages and neutrophils was done in 10 different fields with 200× original magnification in three animals.

Paraffin-embedded lung sections were cut at 5mm, de-paraffinized, rehydrated, and stained as previously described [22 (link)]. Section were labeled with primary rat anti-mouse F4/80 antibodies (Bio-Rad, Portland, ME, USA, 1:20 dilution), and rat anti-mouse neutrophil-specific antibodies (Abcam, Cambridge, MA, USA, 1:200 dilution) following secondary biotinylated goat anti-rat antibodies (Dako North America Inc., Santa Clara, CA, USA). The heat-induced epitope retrieval method was used for both antibodies. The sections were incubated with streptavidin-peroxidase and developed using a 3-amino-9-ethylcarbozole (AEC) kit (both from Thermo Fisher Scientific, Waltham, MA, USA). Sections were counterstained with hematoxylin (Vector Laboratories, Inc., Burlingame, CA, USA). Controls included replacing the primary antibody with equivalent concentrations of the rat non-specific serum. Images were acquired by Olympus IX51 microscope with Olympus DP72 camera (both from Olympus Corporation, Waltham, MA, USA). Quantification of lung infiltrating macrophages and neutrophils was done in ten different fields with 200x original magnification in three animals.