Anti-Nestin antibody [SP103] - Neural Stem Cell Marker

• IHC-Fr

Lab

Immunohistochemistry (Frozen sections) - Anti-Nestin antibody [SP103] - Neural Stem Cell Marker (AB105389)

IHC image of Nestin staining in a section of frozen normal human kidney* performed on a Leica BOND™ system using the standard protocol. The section was fixed in 10% paraformaldehyde (10 min) prior to staining. The section was incubated with ab105389, 5ugml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX. The inset secondary-only control image is taken from an identical assay without primary antibody.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre

• IHC-P

AbReview26930****

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Nestin antibody [SP103] - Neural Stem Cell Marker (AB105389)

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.

This image is courtesy of Hongwei Shao by Abreview.

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Nestin antibody [SP103] - Neural Stem Cell Marker (AB105389)

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

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Nestin antibody [SP103] - Neural Stem Cell Marker (AB105389)

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

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Nestin antibody [SP103] - Neural Stem Cell Marker (AB105389)

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

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-Nestin antibody [SP103] - Neural Stem Cell Marker (AB105389)

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

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Nestin antibody [SP103] - Neural Stem Cell Marker (AB105389)

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

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Nestin antibody [SP103] - Neural Stem Cell Marker (AB105389)

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

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Nestin antibody [SP103] - Neural Stem Cell Marker (AB105389)

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

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Nestin antibody [SP103] - Neural Stem Cell Marker (AB105389)

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

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Nestin antibody [SP103] - Neural Stem Cell Marker (AB105389)

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

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Nestin antibody [SP103] - Neural Stem Cell Marker (AB105389)

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

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Nestin antibody [SP103] - Neural Stem Cell Marker (AB105389)

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.

• IHC-P

PubMed

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Nestin antibody [SP103] - Neural Stem Cell Marker (AB105389)

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)

Malchenko, S. et al PLoS One. 2017 Mar 1;12(3):e0173106. doi: 10.1371/journal.pone.0173106. eCollection 2017 Reproduced under the Creative Commons license http://creativecommons.org/licenses/by/4.0/

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Nestin antibody [SP103] - Neural Stem Cell Marker (AB105389)

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

• WB

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Western blot - Anti-Nestin antibody [SP103] - Neural Stem Cell Marker (AB105389)

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.

All lanes:

Western blot - Anti-Nestin antibody [SP103] - Neural Stem Cell Marker (ab105389) at 1/100 dilution

Lane 1:

Wild-type HAP1 whole cell lysate at 20 µg

Lane 2:

NES knockout HAP1 whole cell lysate at 20 µg

Predicted band size: 177 kDa

false

• WB

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Western blot - Anti-Nestin antibody [SP103] - Neural Stem Cell Marker (AB105389)

All lanes:

Western blot - Anti-Nestin antibody [SP103] - Neural Stem Cell Marker (ab105389) at 1/100 dilution

All lanes:

HeLa cell lysate

Predicted band size: 177 kDa

false