Anti-Musashi 1 / Msi1 antibody [EP1302]

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Musashi 1 / Msi1 antibody [EP1302] (AB52865)

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.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Musashi 1 / Msi1 antibody [EP1302] (AB52865)

ab52865 staining Musashi 1 / Msi1 in HAP1-MSI1 cells. The cells were fixed with 100% methanol (5 min), permeabilized with 0.1% PBS-Tween for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated overnight at 4°C with ab52865 at 1µg/ml and ab7291, Mouse monoclonal [DM1A] to alpha Tubulin - Loading Control. Cells were then incubated with ab150081, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor^® 488), pre-adsorbed at 1/1000 dilution (shown in green) and ab150080, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor^® 594) at 1/1000 dilution (shown in pseudocolour red). Nuclear DNA was labelled with DAPI (shown in blue).

Image was acquired with a confocal microscope (Leica-Microsystems TCS SP8) and a single confocal section is shown.

• Flow Cyt (Intra)

AbReview20152****

Flow Cytometry (Intracellular) - Anti-Musashi 1 / Msi1 antibody [EP1302] (AB52865)

Intracellular Intracellular 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 and permeabilized . The cells were incubated with unpurified ab52865 (1/20 using Prem/wash solution) for 30 mins at 23°C.

This image is courtesy of an Abreview submitted by Jennifer Moore.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Musashi 1 / Msi1 antibody [EP1302] (AB52865)

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.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-Musashi 1 / Msi1 antibody [EP1302] (AB52865)

Intracellular Flow Cytometry analysis of SH-SY5Y (Human neuroblastoma epithelial cell) cells labeling Mushashi 1/ Msi1 with purified ab52865 at 1/80 dilution (10μg/ml) (red). Cells were fixed with 4% Paraformaldehyde and permeabilised with 90% methanol. A Goat anti rabbit IgG (Alexa Fluorr^® 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).

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Musashi 1 / Msi1 antibody [EP1302] (AB52865)

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).

• WB

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Western blot - Anti-Musashi 1 / Msi1 antibody [EP1302] (AB52865)

All lanes:

Western blot - Anti-Musashi 1 / Msi1 antibody [EP1302] (ab52865) at 1/2000 dilution

All lanes:

SH-SY-5Y cell lysate at 10 µg/mL

Secondary

All lanes:

goat anti-rabbit HRP at 1/2000 dilution

Predicted band size: 39 kDa

Observed band size: 39 kDa

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• WB

Lab

Western blot - Anti-Musashi 1 / Msi1 antibody [EP1302] (AB52865)

Blocking and diluting buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-Musashi 1 / Msi1 antibody [EP1302] (ab52865) at 1/1000 dilution

Lane 1:

SH-SY5Y (Human neuroblastoma epithelial cell) whole cell lysates at 20 µg

Lane 2:

UMNSAH/DF-1 (chicken embryo fibroblast) whole cell lysates at 20 µg

Secondary

All lanes:

Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/2000 dilution

Predicted band size: 39 kDa

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• WB

Lab

Western blot - 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.

All lanes:

Western blot - Anti-Musashi 1 / Msi1 antibody [EP1302] (ab52865) at 1/2000 dilution

Lane 1:

Wild-type HAP1 whole cell lysate at 20 µg

Lane 2:

MSI1 knockout HAP1 whole cell lysate at 20 µg

Lane 3:

SH-SY5Y whole cell lysate at 20 µg

Predicted band size: 39 kDa

Observed band size: 39 kDa

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• WB

Lab

Western blot - Anti-Musashi 1 / Msi1 antibody [EP1302] (AB52865)

This blot was produced using 4-20% SDS-PAGE containing 15 μg of SH-SY5Y whole cell lysate per lane at 150V for 1hr before being transferred onto a 0.45 μm PVDF membrane at 75V for 1hr. The membrane was then blocked for 1hr using 5% NFDM/TBST, then incubated with ab52865 (1/1000) at room temperature for 1hr. After being washed three times in TBST, the membrane was incubated with Peroxidase conjugated goat anti-rabbit IgG (H+L) (ab97051) at 1/20,000 for 1hr at room temperature. The membrane was washed three times again. Then the signal was developed using the ECL technique. ab52865 was stored at a range of temperatures (+4°C, +22°C, +37°C) for 1 week before being tested in WB. The image shows the band intensity remains relatively constant across all storage temperatures, demonstrating that antibody activity is not affected.

All lanes:

Western blot - Anti-Musashi 1 / Msi1 antibody [EP1302] (ab52865) at 1/1000 dilution

All lanes:

SH-SY5Y whole cell lysate at 15 µg with NDFM/TBST

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

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Exposure time: 40s

• IHC-P

AbReview9265****

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Musashi 1 / Msi1 antibody [EP1302] (AB52865)

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).

This image is courtesy of an Abreview submitted by Carl Hobbs.