All lanes : Anti-Nestin antibody [SP103] (ab105389) at 1/100 dilution

Lane 1 : Wild-type HAP1 whole cell lysate
Lane 2 : NES knockout HAP1 whole cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 177 kDa

Lanes 1 - 2: Merged signal (red and green). Green - ab105389 observed at 300 kDa. Red - loading control, ab18058, observed at 117 kDa.

ab105389 was shown to specifically react with NES in wild-type HAP1 cells as signal was lost in NES knockout cells. Wild-type and NES knockout samples were subjected to SDS-PAGE. Ab105389 and ab18058 (Mouse anti-Vinculin loading control) were incubated overnight at 4°C at 1/100 dilution and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed ab216776 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

ICC/IF image of ab105389 stained SKNSH cells. The cells were 4% formaldehyde fixed (10 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 (ab105389, 1/200 dilution) overnight at +4°C. The secondary antibody (green) was ab96899, DyLight® 488 goat anti-rabbit 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.

BTIC markers in our CNS-PNET model tumors

Formalin-fixed, paraffin-embedded mouse primitive neural ectodermal tumor tissue was stained for Nestin using ab105389 at 1/30 dilution in immunohistochemical analysis.

Insert x20 magnification.

TU-tumor; P-parenchyma; V-ventricle; CP-choroid plexus

(From Figure 3C of Malchenko et al)

Formalin-fixed, paraffin-embedded human endometrium tissue stained for Nestin using ab105389 at 1/100 dilution in immunohistochemical analysis.

Anti-Nestin antibody [SP103] (ab105389) at 1/100 dilution + HeLa cell lysate

Predicted band size: 177 kDa

Overlay histogram showing SH-SY5Y cells stained with ab105389 (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 (ab105389, 1/50 dilution) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed.

Formalin-fixed, paraffin-embedded human colon tissue stained for Nestin using ab105389 at 1/100 dilution in immunohistochemical analysis.

Formalin-fixed, paraffin-embedded human prostate tissue stained for Nestin using ab105389 at 1/100 dilution in immunohistochemical analysis.

Formalin-fixed, paraffin-embedded human lung tissue stained for Nestin using ab105389 at 1/100 dilution in immunohistochemical analysis.

Formalin-fixed, paraffin-embedded human breast tissue stained for Nestin using ab105389 at 1/100 dilution in immunohistochemical analysis.

Formalin-fixed, paraffin-embedded human skin tissue stained for Nestin using ab105389 at 1/100 dilution in immunohistochemical analysis.

Formalin-fixed, paraffin-embedded human cerebellum tissue stained for Nestin using ab105389 at 1/100 dilution in immunohistochemical analysis.

Formalin-fixed, paraffin-embedded human cervix tissue stained for Nestin using ab105389 at 1/100 dilution in immunohistochemical analysis.

Formalin-fixed, paraffin-embedded human kidney tissue stained for Nestin using ab105389 at 1/100 dilution in immunohistochemical analysis.

Formalin-fixed, paraffin-embedded human ovary tissue stained for Nestin using ab105389 at 1/100 dilution in immunohistochemical analysis.

ab105389 staining Nestin in human melanoma tissue by Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections).
Tissue was fixed in formaldehyde and a heat mediated antigen retrieval step was performed using Tris EDTA pH 9.0. Samples were then permeabilized using 0.25% Triton X-100, blocked, then incubated with ab105389 at a 1/100 dilution for 18 hours at 4°C. The secondary used was an Alexa-Fluor 594 (red) conjugated donkey anti-rabbit polyclonal used at a 1/100 dilution. (Green) tumor cells, (blue) DAPI.

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