Recombinant Anti-CD14 antibody [EPR3652] (ab133503)

Flow cytometry staining of human peripheral blood mononuclear cells (PBMCs) with ab133503 (right) or Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control (left). PBMCs were incubated for 30 min on ice in 1x PBS containing 10 µg/ml human IgG and 10 % normal goat serum to block FC receptors and non-specific protein-protein interaction followed by the antibody ab133503 or Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control (1x 10⁶ in 100 µl at 1.0 μg/ml (1/2300)) for 30min on ice.

The secondary antibody Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed was incubated at 1/4000 for 30min on ice

Acquisition of >30000 events were collected using a 50 mW Blue laser (488nm) and 525/40 bandpass filter. Events were gated on CD3 and CD19 negative cells.

Human whole blood was stained with anti-CD14 antibody ab133503 (red line). In brief, the erythrocytes were lysed and the cells were then stained with anti-CD14 (ab133503)  for 30 min at 4°C. The secondary antibody used was Alexa Fluor® 488 goat anti-rabbit IgG (H&L) (ab150077) at 1/2000 dilution for 30 min at 4°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (ab172730) used at the same concentration and conditions as the primary antibody. Unlabelled sample (blue line) was also used as a control.

Acquisition of >30,000 total events were collected. Gating strategy - peripheral blood monocytes

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human tonsil tissue labelling CD14 with purified ab133503 at 1/500. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human colon tissue labelling CD14 with unpurified ab133503 at a dilution of 1/100.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human tonsil tissue labelling CD14 with unpurified ab133503 at a dilution of 1/100.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.