Rabbit anti neutrophil elastase antibody

FITC-labeled M. avium (FITC-Mav) was prepared and bacterial localization by confocal microscopy was performed as previously described (50 (link)). FITC-Mav were sonicated, washed, and resuspended into 0.5 mL of PBS. FITC-Mav was combined with neutrophils for 1 h at a multiplicity of infection of approximately 5:1. The cells were cytocentrifuged to microscope slides and then air-dried. The slides were processed with rabbit anti-neutrophil elastase antibody (Abcam, Boston, MA), 4′,6-diamidino-2-phenylindole (DAPI), and Alexa Fluor 555-conjugated goat anti-rabbit (Invitrogen, Waltham, MA) and imaged using a Zeiss Axiovert 200M microscope. Images were quantified by calculating percent FITC(+) cells per field.

The following primary antibodies and secondary antibodies have been used: mouse monoclonal anti-Ki67 antibody (BD Biosciences, San Diego, CA, USA; 1:200); mouse monoclonal anti-CagA antibody (A-10) (1:200), rabbit polyclonal anti-CagA antibody (b-300) (1:200), rabbit anti-E-cadherin polyclonal antibody (H-108) (1:200), mouse anti-E-cadherin monoclonal antibody (G-10) (1:200) (All above antibodies were purchased from Santa Cruz Biotechnology Inc, Santa Cruz, California, USA); rabbit H. pylori polyclonal antibody (prediluted, ready to use), rabbit anti-Ki67 antibody (1:100) (Maixin Bio., Fuzhou, China); rabbit anti-MPO antibody (1:200), mouse anti-MPO antibody (1:200), rabbit anti-MMP9 antibody (1:200), rabbit anti-beta-Catenin antibody (1:200) (PTGLabs, Chicago, IL, USA); rabbit anti-Neutrophil elastase antibody (Abcam, Cambridge, MA,USA; 1:200); donkey anti-mouse Alexa Fluor 594 secondary antibody, donkey anti-rabbit Alexa Fluor 594 secondary antibody and donkey anti-rabbit Alexa Fluor 488 secondary antibody (Jackson ImmunoResearch, West Grove, PA, USA; 1:400).

Paraffin sections (5 μm) of organs from infected or control animals were deparaffinized and subjected to antigen retrieval by boiling in 10 mM citrate buffer at pH 6.0 before equilibration in PBS and blocking in PBS with 5% BSA (AppliChem), 5% goat serum (Sigma-Aldrich), and 0.5% Fc-block (Becton, Dickinson) were performed. Neutrophils were stained with polyclonal rabbit anti-neutrophil elastase antibody (Abcam, Inc.) followed by goat anti-rabbit Cy3 (Jackson ImmunoResearch). N. meningitidis was then stained using fluorescein isothiocyanate (FITC)-labeled anti-N. meningitidis polyclonal rabbit antibody. The rabbit anti-N. meningitidis antibody raised against strain MC58 was described elsewhere (74 (link)). Sections were counterstained with DAPI (4′,6-diamidino-2-phenylindole) and mounted with Fluoroshield (Sigma-Aldrich). Immunofluorescence microscopy was carried out on a BZ-9000 Biorevo digital microscope (Keyence) at ×60 and ×100 magnification using the full-focus function to collapse z-stacks in BZ II Viewer software for image capture and the black balance function and channel merge function in BZ II Analyzer software.