Overlay histogram showing SH-SY5Y cells stained with JP104234 (green line). The cells were fixed with 2% paraformaldehyde (10 min) and then permeabilized with 90% methanol for 10 min. The cells were then icubated in 2% bovine serum albumin to block non-specific protein-protein interactions followed by the antibody (JP104234, 1:25 dilution) for 60 min at 37ºC. The secondary antibody used was Goat-Anti-Rabbit IgG, DyLight® 488 Conjugated Highly Cross-Adsorbed(NA168821) at 1/400 dilution for 40 min at 37ºC. Isotype control antibody (blue line) was rabbit IgG (1μg/1×10^6 cells) used under the same conditions. Acquisition of >10, 000 events was performed.

JP104234 staining Nestin in Human heart tissue sections by Immunohistochemistry (IHC-P – paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with formaldehyde and blocked with 3% BSA for 0. 5 hour at room temperature; antigen retrieval was by heat mediation with a citrate buffer (pH6). Samples were incubated with primary antibody (1/25) for 1 hours at 37°C. A undiluted biotinylated goat polyvalent antibody was used as the secondary antibody.

Anti-Nestin Antibody (S1409) at 1:2000 dilution + Hela whole cell lysateLysates/proteins at 20 µg per lane. SecondaryGoat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/10000 dilution. Predicted band size : 177 kDaBlocking/Dilution buffer: 5% NFDM/TBST.

Anti-Nestin Antibody (S1409)at 1:2000 dilution + Hela whole cell lysatesLysates/proteins at 20 µg per lane. SecondaryGoat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/10000 dilutionPredicted band size : 177 kDaBlocking/Dilution buffer: 5% NFDM/TBST.

Fluorescent confocal image of SY5Y cells stained with Nestin (S1409) antibody. SY5Y cells were fixed with 4% PFA (20 min), permeabilized with Triton X-100 (0.2%, 30 min). Cells were then incubated with JP104234 Nestin (S1409) primary antibody (1:200, 2 h at room temperature). For secondary antibody, Alexa Fluor® 488 conjugated donkey anti-rabbit antibody (green) was used (1:1000, 1h). Nuclei were counterstained with Hoechst 33342 (blue) (10 μg/ml, 5 min). Note the highly specific localization of the Nestin (S1409) immunosignal to the intermediate filaments, supported by Human Protein Atlas Data (http://www.proteinatlas.org/ENSG00000132688).

Western blot analysis of Nestin Antibody (S1409) (Cat.#JP104234) in HepG2 cell line lysates (35ug/lane). NES (arrow) was detected using the purified Pab.

Formalin-fixed and paraffin-embedded human hepatocarcinoma reacted with Nestin Antibody (S1409), which was peroxidase-conjugated to the secondary antibody, followed by DAB staining. This data demonstrates the use of this antibody for immunohistochemistry; clinical relevance has not been evaluated.