Dylight 488 conjugated goat anti rabbit

Immunofluorescent staining for nestin was performed according to a standard protocol (31 ). Briefly, the cells were incubated with rabbit anti-nestin polyclonal antibody (1:100; AB5922; EMD Millipore, Billerica, MA, USA), followed by incubation with cyanine 2-conjugated goat anti-rabbit (1:300; 111-225-144) or DyLight 488-conjugated goat anti-rabbit (1:400; 111-545-003) antibodies (both Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA). Cells were then observed under a fluorescent microscope.

The antibodies used were rat anti‐CD169 (AbD Serotec, MCA‐884; 1:200), rabbit anti‐iba1 (Waco, 019‐19741; 1:200) and rabbit anti‐HO‐1 (Abcam, ab13243; 1:250). The secondary antibodies were biotin conj. goat anti‐rat (Amersham, RPN 1005; 1:1500), followed by Vectastain ABC reagent Vector (PK‐7100) and DAB (DakoCytomation), using the companies' recommendations. For rabbit antibodies, EnVision (DakoCytomation) was used. For double‐staining RH RedX‐conjugated goat anti‐rat (Jackson, 112‐295‐102) and Dylight 488‐conjugated goat anti‐rabbit (Jackson, 111‐485.144) were used. Specimens incubated with rat IgG2a (AbD Serotec), irrelevant rabbit antibodies or no primary antibodies served as controls.