Musashi 1

Manufactured by Cell Signaling Technology

Sourced in United States

Musashi-1 is a protein involved in the regulation of gene expression and cell fate determination. It is a highly conserved RNA-binding protein that plays a crucial role in stem cell maintenance and differentiation. Musashi-1 is expressed in various types of stem and progenitor cells and is commonly used as a marker for these cell populations.

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4 protocols using musashi 1

Stem Cell Marker Expression Analysis

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To evaluate stem cell marker expression, neurospheres were dissociated mechanically or enzymatically with Accutase (Gemini Bioscience, Sacramento, CA). To facilitate adherence, cells were plated on poly-L-lysine/laminin coated four-well plates in neurosphere media. Cells were fixed in 4% paraformaldehyde, blocked and permeabilized with a 5% bovine serum albumin (BSA) with 0.6% Triton-× 100 and then treated with the primary antibodies Nestin (Abcam, Cambridge, MA), Sox2, Musashi 1, CD44, Bmi-1 (Cell Signaling Technology, Danvers, MA), CD133 (Biorbyt, Cambridge, UK) and A2B5 (A2B5 clone 105, ATCC, Manassas, VA). A “no primary control” was included for all antibodies tested for all cell lines. For these, the cells were incubated with only the antibody diluent (2.5% BSA, 0.3% triton, balance PBS). Cells were then treated with a fluorochrome-conjugated secondary antibody followed by Prolong Gold Antifade Reagent with DAPI (Thermo Fisher Scientific, Waltham, MA). Samples were examined under an EVOS FLoid Cell Imaging Station fluorescent microscope (Thermo Fisher Scientific, Waltham, MA).

Gersey Z.C., Rodriguez G.A., Barbarite E., Sanchez A., Walters W.M., Ohaeto K.C., Komotar R.J, & Graham R.M. (2017). Curcumin decreases malignant characteristics of glioblastoma stem cells via induction of reactive oxygen species. BMC Cancer, 17, 99.

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Western Blot Analysis of Protein Levels

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Total cell lysates were prepared in NP-40 lysis buffer supplemented with 1 mM DTT, 0.1 mM PMSF and protease inhibitors [48 (link)]. Protein samples (30 μg of total lysates) were denatured and resolved on 10% NuPage gels (Life Technologies) and transferred onto Protran NC membrane (IscBioExpress). Western blots were dried at room temperature for 1 h and blocked with 1% BSA in TBST for 1 h. Primary antibody in 1% BSA and TBST was typically incubated overnight at 4°C (dilution 1:1000), washed, incubated with Anti-rabbit or Anti-mouse IgG HRP conjugate secondary antibody (Promega), and exposed using ChemiGlow substrate on the ChemiImager Imaging System (Alpha Innotech). Antibodies were obtained from Abcam (Musashi-1), Cell Signaling (GAPDH), Millipore (p21) and Sigma (α-tubulin). The generation and characterization of the phospho-specific Musashi1 S337 antibody has been described previously [31 (link)]. Relative protein levels were quantitated as previously described [49 (link)-51 (link)] using AlphaEaseFC software (Alpha Innotech). Representative western blots are shown.

MacNicol A.M., Hardy L.L., Spencer H.J, & MacNicol M.C. (2015). Neural stem and progenitor cell fate transition requires regulation of Musashi1 function. BMC Developmental Biology, 15, 15.

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Cerebral Organoid Immunohistochemistry Protocol

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Cerebral organoids were fixed in 4% paraformaldehyde for 45 minutes at room temperature followed by three PBS washes for 10 minutes each. Samples were incubated in 30% sucrose overnight and embedded in OCT for cryosectioning. Frozen sections (10 μm thickness) were stained with hematoxylin and eosin or used for immunostaining. Sections were blocked and permeabilized in 0.3% Triton X-100 and 3% normal goat serum in PBS. The following primary antibodies were used: N-cadherin (Cell Signaling, 13116S, 1:200), SOX2 (Cell Signaling, 3579S, 1:200), TUJ1 (BioLegend, 845502, 1:200), PAX6 (Santa Cruz, sc-81649, 1:200), TBR2 (Abcam, ab23345, 1:200), MAP2 (Cell Signaling, 4542S, 1:200), Musashi-1 (Cell Signaling, 85652S, 1:200), Nestin (BioLegend, 841801, 1:200), and GFAP (IHC: Cell Signaling, 12389S, 1:200; IF: Dako, ZO334, 1:1000). Secondary antibodies used were goat Alexa Fluor 488 and 594 conjugates (Thermo Fisher Scientific, 1:500). For immunofluorescence, DAPI (4, 6-diamidino-2 -phenylindole, dihydrochloride) was used as a counterstain at a concentration of 1.0 μg/mL in PBS. Histopathologic evaluation was performed by board-certified neuropathologist, Dr. Matija Snuderl (NYU Langone Medical Center).

Linkous A., Balamatsias D., Snuderl M., Edwards L., Miyaguchi K., Milner T., Reich B., Cohen-Gould L., Storaska A., Nakayama Y., Schenkein E., Singhania R., Cirigliano S., Magdeldin T., Lin Y., Nanjangud G., Chadalavada K., Pisapia D., Liston C, & Fine H.A. (2019). Modeling Patient-Derived Glioblastoma with Cerebral Organoids. Cell reports, 26(12), 3203-3211.e5.

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Western Blot and IHC Antibody Sources

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Primary antibodies for Western blotting and immunohistochemistry were purchased as follows: β-actin, Lamin B, cyclooxygenase (COX-2), nitric oxide synthase (iNOS), p53, cytochrome c, Survivin, epidermal growth factor receptor (EGFR), Ki-67 and cluster of differentiation 31 (CD31) antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA), phosphorylated signal transducer and activator of transcription 3 (STAT3), γ-H2AX, Bax, B-cell lymphoma 2 (Bcl-2), cleaved caspase-3, cleaved caspase-8, poly (ADP-ribose) polymerase (PARP), lysozyme and Musashi-1 from Cell Signaling Technology, Inc.(Danvers, MA, USA).

Han Y.M., Park J.M., Choi Y.S., Jin H., Lee Y.S., Han N.Y., Lee H, & Hahm K.B. (2017). The efficacy of human placenta-derived mesenchymal stem cells on radiation enteropathy along with proteomic biomarkers predicting a favorable response. Stem Cell Research & Therapy, 8, 105.

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