Biotinylated mouse anti rat igg

Phospho-ERK staining was markedly increased in cells lining Bowman's capsule in this FSGS model in other mouse strains.⁴⁴ Triple immunofluorescence was performed using the following primary antibodies: rat monoclonal antibody to CD44 (1:10, BD Biosciences, San Jose, CA, USA), rabbit polyclonal antibody to phospho-p44/42 MAPK (1:250, Cell Signaling Technology, Beverly, MA, USA), anti-NPHS2 and anti-β-gal as described above. P-p44/42 (to identify phosphorylated ERK 1/2) was used with an Alexa647 conjugated donkey anti-rabbit IgG-(1:100, Jackson, West Grove, PA, USA). Anti-NPHS2 was visualized with AlexaFluor488 conjugated goat anti-rabbit IgG- (1:100, Invitrogen, Eugene, OR, USA). The secondary antibody for CD44 was biotinylated mouse anti-rat IgG (1:200, Jackson ImmunoResearch, West Grove, PA), visualized with either Alexa488 or -Alexa647 conjugated streptavidin (1:100, Life Technologies, Eugene, OR, USA). Cells in these sections were considered double- or triple-positive using signal overlap in the cytoplasm.