Rat anti mouse gr 1 rb6.8c5 fitc

Frozen renal cortex sections (2 μm) were fixed in ice-cold acetone for 10 minutes and incubated with primary antibodies diluted in PBS containing 1% bovine serum albumin and 0.05% sodium azide (IF-buffer) for 60 minutes. Directly labeled antibodies included goat anti-mouse C3c- and fibrinogen-fluorescein isothiocyanate (FITC) (Nordic, Tilburg, The Netherlands), goat anti-rabbit IgG Alexa-488, donkey anti-goat IgG Alexa-594 (Life technologies, Breda, The Netherlands), goat anti-Armenian hamster IgG Cy3 (Jackson ImmunoResearch Laboratories, West Grove, PA) and rat anti-mouse GR-1 (RB6.8C5)-FITC (BD Biosciences, Alphen aan de Rijn, The Netherlands). Unlabeled primary antibodies included CD68 (MCA1957; Serotec, Oxford, UK), goat anti-VSV-G protein (Novus Biologicals, Cambridge, UK) and hamster anti-mouse Agrin (MI91) (Dept. of Nephrology, Nijmegen, NL). Sections were postfixed with 1% paraformaldehyde in PBS and embedded in VectaShield mounting medium H-1000 (Brunschwig Chemie, Amsterdam, The Netherlands). Goat anti-rabbit IgG, goat anti-mouse C3c, and fibrinogen staining intensities were scored semi-quantitatively on blinded sections from 0 (no staining) to 10 (100% staining intensity inside the glomeruli) independently by two researchers and averaged over 50 glomeruli. Glomerular influx of PMNs was quantified by counting the number of cells per 50 glomeruli.

Frozen sections (2 µm) were fixed in ice-cold acetone for 10 min and stained essentially as described previously (Rops et al., 2007b (link)). Directly labeled antibodies included goat anti-mouse C3c and fibrinogen-fluorescein isothiocyanate (FITC) (Nordic, Tilburg, Netherlands), goat anti-rabbit IgG Alexa-488 (Life Technologies, Breda, Netherlands), rat anti-mouse GR-1 (RB6.8C5)-FITC (BD Biosciences, Alphen aan de Rijn, Netherlands), and goat anti-Armenian hamster-Cy3 (Jackson ImmunoResearch Laboratories, West Grove, PA). Unlabeled primary antibodies included rat anti-mouse-CD68 (MCA 1957; Serotec, Oxford, United Kingdom) and hamster anti-agrin (MI91) (Raats et al., 1998 (link)). Sections were fixed with 1% paraformaldehyde–PBS and embedded in VectaShield mounting medium H-1000 (Brunschwig Chemie, Amsterdam, Netherlands). Goat anti-rabbit IgG, goat anti-mouse C3c, fibrinogen, and anti-HS scFv staining intensities were evaluated semi-qualitatively from 0 (no staining) to 10 (100% staining intensity inside the glomeruli) and averaged over 50 glomeruli. All quantitative observations were made by two independent observers on blinded sections. Glomerular influx of granulocytes was determined by counting the number of cells per 50 glomeruli.