Anti mouse igg alexa488 antibody

Fluorescence immunohistochemistry was performed as previously described(20 (link)). Five μm sections were cut from the paraffin blocks and antigen retrieval was accomplished by heating in 1mM EDTA, pH 9.0, for 10min at 90°C. Samples were cooled to room temperature and blocked with 5% BSA in TBST for 5min at room temperature. A rabbit anti-EGFR antibody (ab2430, abcam, Cambridge, MA) was used to characterize EGFR expression and a mouse anti-factor VIII (ab139391, abcam, Cambridge, MA) was used to characterize vessel density via factor VIII. Primary antibodies were applied at the recommended concentrations and incubated overnight at 4°C. After washing, an anti-rabbit IgG-Alexa 546 antibody (A-11035, Life Technologies, Carlsbad, CA) was applied followed by an anti-mouse IgG-Alexa 488 antibody (A-11001, Life Technologies, Carlsbad, CA) at manufacturer recommendation. After washing, slides were imaged by a blinded operator (J. Kovar) using an Olympus IX81 Inverted Microscope equipped with a halogen bulb and following filters (Alexa 488: EX 488/20, DC495LP; EM 525/50; Alexa 546: EXET545/30x, T570LP, EM ET620/60M; IRDye800: EX: HQ760/40x, 790DCXR, EM: HQ830/50m; Chroma Technology Corp., Rockingham, VT).