Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human spleen tissue sections labeling Nestin with purified ab176571 at 1:1000 dilution (1.65 µg/ml). Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0). ImmunoHistoProbe one step HRP Polymer (ready to use)was used as the secondary antibody. Negative control:PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human kidney tissue sections labeling Nestin with purified ab176571 at 1:1000 dilution (1.65 µg/ml). Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0). ImmunoHistoProbe one step HRP Polymer (ready to use)was used as the secondary antibody. Negative control:PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

Immunocytochemistry/ Immunofluorescence analysis of U-87 MG (Human glioblastoma-astrocytoma epithelial cell) cells labeling Nestin with purified ab176571 at 1:150 dilution (10 µg/ml). Cells were fixed in 100% Methanol and permeabilized with None. Cells were counterstained with Ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) 1:200 (2.5 µg/ml). Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) was used as the secondary antibody at 1:1000 (2 µg/ml) dilution. DAPI nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

ICC/IF image of ab176571 stained SK-N-SH (Human neuroblastoma cell line) cells.

The cells were fixed in 100% methanol (5 min) and then incubated in 1% BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilize the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody ab176571 at 5 µg/ml overnight at +4°C. The secondary antibody (pseudo-colored green) was Alexa Fluor^® 488 goat anti-rabbit (ab150081) IgG (H+L) preadsorbed, used at a 1/1000 dilution for 1h. Alexa Fluor^® 594 WGA was used to label plasma membranes (pseudo-colored red) at a 1/200 dilution for 1h at room temperature. DAPI was used to stain the cell nuclei (pseudo-colored blue) at a concentration of 1.43 µM for 1hour at room temperature.

Immunohistochemical analysis of paraffin-embedded human melanoma tissue labeling Nestin using ab176571 at a 1/250 dilution.

Heat mediated antigen retrieval was performed with citrate buffer pH 6 before commencing with IHC staining protocol.

Flow Cytometry analysis of SH-SY5Y (human neuroblastoma) cells labeling Nestin with purified ab176571 at 1/160 dilution (10 µg/ml) (red). Cells were fixed with 4% paraformaldehyde and permeabilized with 90% methanol. A Goat anti rabbit IgG (Alexa Fluor^® 488)(1/2000 dilution) was used as the secondary antibody. Rabbit monoclonal IgG (Black) was used as the isotype control, cells without incubation with primary antibody and secondary antibody (Blue) were used as the unlabeled control.

Immunohistochemical analysis of paraffin-embedded human kidney tissue labeling Nestin using ab176571 at a 1/250 dilution.

Heat mediated antigen retrieval was performed with citrate buffer pH 6 before commencing with IHC staining protocol.