Immunocytochemistry/ Immunofluorescence analysis of CTC isolated from mRCC patients stained for hematopoietic cells labeling CD45 with ab8216 (green). The cells were fixed in 4.5% paraformaldehyde for 15 min, washed in PBS, permeabilized with 1x Perm/Wash for 10 min, washing in PBS, blocking of unspecific antibody reactions by incubation with blocking solution containing 5% BSA for 30 min, binding of Anti-CD45 antibody [MEM-28] (ab8216) (final concentration: 5 μg/ml) overnight at 4°C, wash in 0,1% Tween, binding of secondary antibody (Cy3-conjugated goat anti-mouse) for 30 min at 37°C, washing in 0,1% Tween. Subsequently, cells were stained with DAPI for 10 min, mounted with anti-fading medium and stored in the dark until evaluation. Left: CD45, right: DAPI, bottom: merge.

Ab8216 staining human normal tonsil tissue. Staining is localised to cellular membranes.
Left panel: with primary antibody at 1 ug/ml. Right panel: isotype control.
Sections were stained using an automated system (DAKO Autostainer Plus ), at room temperature: sections were rehydrated and antigen retrieved with the Dako 3 in 1 AR buffers citrate pH6.1 in a DAKO PT Link. Slides were peroxidase blocked in 3% H2O2 in methanol for 10 mins. They were then blocked with Dako Protein block for 10 minutes (containing casein 0.25% in PBS) then incubated with primary antibody for 20 min and detected with Dako envision flex amplification kit for 30 minutes. Colorimetric detection was completed with Diaminobenzidine for 5 minutes. Slides were counterstained with Haematoxylin and coverslipped under DePeX. Please note that for manual staining we recommend to optimize the primary antibody concentration and incubation time (overnight incubation), and amplification may be required.

Flow cytometry analysis (surface staining) of human peripheral blood cells with ab8216 (1 ug/ml), GAM-APC.

Immunocytochemistry/ Immunofluorescence analysis of citrated peripheral blood hematologic cells taken from head and neck squamous cell carcinoma (HNSCC) patients labeling CD45 with ab8216 (green). The staining method included fixation of the cells in 4.5% paraformaldehyde for 15 min, washing in PBS, permeabilization with 1× Perm/Wash Buffer for 10 min, washing in PBS, blocking of unspecific antibody reactions by incubation with blocking solution containing 5% BSA for 30 min, binding of Anti-CD45 antibody [MEM-28] (ab8216) (final concentration: 5 µg/ml) overnight at 4°C, wash in 0,1% Tween, binding of Cy3-conjugated secondary antibody for 30 min at 37°C, washing in 0,1% Tween.

Immunocytochemistry/ Immunofluorescence analysis of hematopoietic cells labeling CD45 with ab8216. The cells were fixed in 4.5% paraformaldehyde for 15 min, washed in PBS, permeabilized with 1x Perm/Wash for 10 min, washing in PBS, blocking of unspecific antibody reactions by incubation with blocking solution containing 5% BSA for 30 min, binding of Anti-CD45 antibody [MEM-28] (ab8216) (final concentration: 5 μg/ml) overnight at 4°C, wash in 0,1% Tween, binding of secondary antibody (Cy3-conjugated goat anti-mouse) for 30 min at 37°C, washing in 0,1% Tween.

Overlay histogram showing peripheral blood lymphocytes stained with ab8216 (red line). The cells were incubated with the antibody (ab8216, 1µg/1x10⁶ cells) for 30 min at 4ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H&L) (ab96879) at 1/200 dilution for 30 min at 4ºC. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed gating on peripheral blood lymphocytes.

Immunocytochemistry/Immunofluorescence analysis of human peripheral blood mononuclear cells labelling CD45 (green) with ab8216 at 10 μg/mL. Nuclei were counterstained with DAPI (blue).