Recombinant Anti-Musashi 1 / Msi1 antibody [EP1302] (ab52865)

Lanes 1 - 3: Merged signal (red and green). Green - ab52865 observed at 39 kDa. Red - loading control, ab130007, observed at 130 kDa.

ab52865 was shown to specifically react with Musashi 1 / Msi1 in wild-type HAP1 cells as signal was lost in MSI1 knockout cells. Wild-type and MSI1 knockout samples were subjected to SDS-PAGE. Ab52865 and ab130007 (Mouse anti Vinculin loading control) were incubated overnight at 4°C at 1/2000 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.

Blocking and diluting buffer: 5% NFDM/TBST.

Flow Cytometry analysis of SH-SY5Y (Human neuroblastoma epithelial cell) cells labeling Mushashi 1/ Msi1 with purified ab52865 at 1:80 dilution (10 µg/ml) (red). Cells were fixed with 4% Paraformaldehyde and permeabilised with 90% methanol. A Goat anti rabbit IgG (Alexa Fluor^® 488) secondary antibody was used at 1:2000 dilution. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue).

Immunocytochemistry/ Immunofluorescence analysis of SH-SY5Y (Human neuroblastoma epithelial cell) cells labeling Mushashi 1/ Msi1 with Purified ab52865 at 1:500 dilution. Cells were fixed in 4% Paraformaldehyde and permeabilized with 0.1% tritonX-100. Cells were counterstained with Ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) 1:200. Ab150077 Goat anti rabbit IgG(Alexa Fluor^® 488) was used as the secondary antibody at 1:1000 dilution. DAPI nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human lung carcinoma tissue sections labeling Mushashi 1/ Msi1 with Purified ab52865 at 1:50 dilution (17.7 μg/ml). Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0). Tissue was counterstained with Hematoxylin. ab97051 Goat Anti-Rabbit IgG H&L (HRP) secondary antibody was used at 1:500 dilution. PBS instead of the primary antibody was used as the negative control.

Immunocytochemistry/Immunofluorescence analysis of Neuro-2a (mouse neuroblastoma) labelling Musashi 1 with purified ab52865 at 1/500. Cells were fixed with 4% Paraformaldehyde and permeabilised by 0.1% tritonX-100. An Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody (Ab150077). Nuclei counterstained with DAPI (blue).

Immunohistochemistical detection (on formaldehyde/PFA-fixed paraffin-embedded sections) of Musashi 1 / Msi1 antibody [EP1302] (unpurified ab52865) on Quail Tissue sections (embryo d5/6 Brain stem T/S). Antigen retrieval step: Heat mediated. Blocking step: 1% BSA for 10 mins at RT. Primary Antibody unpurified ab52865 incubated at 1/300 for 2 hours at RT. Secondary Antibody: Biotin labelled goat anti rabbit IgG (1/300).

Flow Cyt image of Musashi1 (ab52865)using Accutase digested single cell suspension of hESC. the cells were fixed adn permeabilized. The cells were incubated with unpurified ab52865 (1/20 using Prem/wash solution) for 30 mins at 23°C.

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