Immunocytochemistry analysis of RAW264.7 cells untreated / treated with 100nM TPA overnight then 100ng/mL LPS for 7 hours add 1μg/mL BFA for last 3 hours labeling CCL2 / MCP-1 with Rabbit anti-CCL2 / MCP-1 antibody (HA500267) at 1/500 dilution. Cells were fixed in 4% paraformaldehyde for 15 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 15 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-CCL2 / MCP-1 antibody (HA500267) at 1/500 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI. Beta tubulin (HA601187, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution.

Flow cytometric analysis of RAW264.7 cells untreated (left) / treated with 100nM TPA overnight then 100ng/mL LPS for 7 hours add 1μg/mL BFA for last 3 hours (right) labeling CCL2 / MCP-1. Cells were fixed and permeabilized. Then stained with the primary antibody (HA500267, 1/1,000) (red) compared with Rabbit IgG Isotype Control (green). After incubation of the primary antibody at +4℃ for an hour, the cells were stained with a iFluor™ 488 conjugate-Goat anti-Rabbit IgG Secondary antibody (HA1121) at 1/1,000 dilution for 30 minutes at +4℃. Unlabelled sample was used as a control (cells without incubation with primary antibody; black).