A2B5+ cultures were stained with anti‐O4 (1:50; IgM purified from hybridoma culture) for 30 min prior to fixation with 4% PFA for 20 min at room temperature followed by goat antimouse IgM Cy3 (Millipore; 1:200). Murine neurospheres were fixed in 4% PFA for 20 min at room temperature followed by rabbit anti‐NG2 (Millipore; 1:500) and mouse anti‐GFAP (Sigma; 1:1000) for 1 h at room temperature. Cells were incubated with appropriate secondary antibodies (Sigma; 1:1000) for 1 h at room temperature. DAPI (1:1000) was used as a nuclear counterstain for 5 min. All slides were imaged by fluorescence microscopy and quantified by a blinded observer. Cells for each donor/biological replicate were plated in duplicate, and multiple fields of view (6–8 per biological replicate) were imaged. Quantification was performed using manual (NG2+ and O4+ cells) and automatic (DAPI+ Nuclei) counter plugins within the ImageJ (imagej.nih.gov/ij/) software.
Goat antimouse igm cy3
Manufactured by Merck Group
Goat antimouse IgM Cy3 is a reagent used in immunological research applications. It is a conjugate of goat-derived antibodies specific to mouse immunoglobulin M (IgM) and the fluorescent dye Cyanine 3 (Cy3). This reagent can be used to detect and visualize the presence of mouse IgM in biological samples.
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2 protocols using goat antimouse igm cy3
1
Quantifying Oligodendrocyte Differentiation
2
Immunostaining of Oligodendrocyte Lineage
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O4 immunostaining was performed on live, unfixed cells. All other immunostaining was performed after cells were fixed with 4% paraformaldehyde. O4 monoclonal antibodies (1:20, grown from hybridoma cells in our lab; Knapp and Hauser, 1996 (link)) were applied at room temperature for 15 min. Goat anti-mouse IgM-Cy3 (1:1000, Millipore, Billerica, MA) was used to probe bound O4 antibodies. All LPA receptor antibodies (Anti- LPAR1 (Abcam), LPAR2, and LPAR3; Santa Cruz, Dallas, TX; LPAR4, Alomone labs, Hadassah Ein Kerem, Israel; and LPAR6, Acris, San Diego, CA) were applied at 1:1000 at room temperature for 1 hr, and corresponding secondary antibodies were conjugated to Alexa 488 (1:2000, Life Technologies). Antibody specific to ATX (Cosmo Bio, Japan) was also used at 1:1000 at room temperature. The cell nucleus was stained with Hoechst 33342 dye (1:2000, Life Technologies). Images were taken on a confocal microscope (Zeiss LSM 700, Carl Zeiss, Thornwood, NY) and processed using Zeiss Zen 2010 software. OLG morphology was assessed by determining the overall process network (total O4+ area minus the area occupied by the cell body) as described previously (Dennis et al., 2008 (link)), but using ImageJ software (NIH). Evaluators were blinded to treatment groups.
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