Leitz dm irb microscope

PMN detection in the lung tissue was performed by using a Vectastain ABC kit (Vector Laboratories; Linaris; Dossenheim; Germany). Sections were blocked with avidin solution (Vector Laboratories; Burlingame, CA, USA) for one hour to avoid unspecific binding sites and followed by incubation with a rat anti-mouse Ly6G antibody (clone RB6-8C5; ab25377; Abcam; Cambridge, UK). Sections were incubated with biotinylated anti-rabbit IgG (BA-4000; Vector Laboratories; Germany) for one hour, followed by Vectastain ABC reagent (PK-4000; Vector Laboratories; Germany) for 30 min and then incubated with DAB substrate. Nuclear fast red (Linaris; Germany) was used for tissue counterstaining. Rat IgG was utilized as a control (sc-2026; Santa Cruz Biotechnology; Dallas, TX, USA). Tissue slides were processed with a Leitz DM IRB microscope (Leica; Wetzlar, Germany) and analyzed with AxioVision v4.8.2 (Zeiss microscopy; Jena, Germany). PMN counts were enumerated from four random sections in each group by independent observers and reported as number of PMNs per high power field (HPF) as described before [40 (link)].

Immunohistochemistry of neutrophils in lung sections was performed with the Vectastain ABC Kit (Linaris, Mannheim, Germany). After blocking with avidin blocking solution (Vector Labs, Burlingame, USA) for 1 h at room temperature, sections were incubated with a monoclonal rat Ly-6B.2 Alloantigen Antibody 7/4 (AbD Serotec, Cat. No. MCA771GA) or rat IgG control (Santa Cruz, Cat. No. sc-2026) at a dilution of 1:1000 overnight at 4°C. Tissue sections were then incubated with biotinylated rabbit anti-rat IgG (Vector Labs, Cat. No. BA-4000) for 30 min followed by Vectastain ABC reagent (Vector Labs, Cat. No. PK-4000) for 30 min and then developed using Histogreen (Liniaris, Cat. No. E109) as substrate. Counterstaining was performed using nuclear fast red (Linaris, Cat. No. H-3403). Images were then processed with a Leitz DM IRB microscope (Leica). All images were analyzed with the software AxioVision v4.8.2

PMN accumulation in peritoneal tissue, lung and liver sections was visualized via immunohistochemistry by using a Vectastain ABC kit (PK-4000; Vector Laboratories; Germany). Sections were blocked with Avidin solution (Vector Laboratories; Germany) for 1 h to avoid unspecific binding sites. PMNs were stained with rabbit anti-mouse Ly-6G (clone 1A8; Abcam; UK). Rabbit IgG was used as control (31235; Invitrogen; USA). Sections were incubated with biotinylated anti-rabbit IgG (BA-4000; Vector Laboratories; USA) for 1 h, followed by Vectastain ABC reagent (PK-4000; Vector Laboratories; USA) for 30 min and then incubated with DAB substrate. Nuclear fast red (H-3403; Linaris; Germany) was used for tissue counterstaining. Tissue slides were processed with a Leitz DM IRB microscope (Leica) and analyzed with AxioVision v4.8.2 (Carl Zeiss MicroImaging; Germany). Neutrophil counts were examined by enumerating the positive and therefore brown stained cells in a masked fashion. PMN numbers were scored from four random sections of four different tissue samples in each group by two independent observers (41 (link)).