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 (Neural stem cells derived from human embryonic). 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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