ICC/IF image of ab56297 stained RAW246.7 cells. The cells were 100% methanol fixed (5 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab56297, 10µg/ml) overnight at +4°C. The secondary antibody (green) was ab96887, DyLight® 488 goat anti-rat IgG (H+L) used at a 1/250 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM

Bouin’s solution fixed and paraffin embedded mouse kidney section from anti-GBM model was subjected to immunohistochemistry staining (ABC) of Macrophage using ab56297.

Ab56297 staining macrophage in mouse brain tumour tissue sections by (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with paraformaldehyde and blocked with 0.5% TNB blocking reagent for 30 minutes at 25°C. Samples were incubated with primary antibody at 1/100 dilution for 18 hours at 4 C. A goat anti-rat IgG H&L (HRP) (ab7097) was used at 1/500 dilution.

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Overlay histogram showing RAW 264.7 cells stained with ab56297 (red line). The cells were fixed with 4% paraformaldehyde (10 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab56297, 2µg/1x10⁶ cells) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-rat IgG (H+L) (ab98386) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was rat IgG2a, kappa monoclonal [aRTK2758] (ab18450, 2µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in RAW 264.7 cells fixed with 80% methanol/permeabilized in 0.1% PBS-Tween used under the same conditions.

Immunohistochemical analysis of murine tumour tissue, staining Macrophage with ab56297. Antigen retrieval was performed under high pressure in 10 mM EDTA buffer (pH 8.0) before incubation with primary antibody.