Multiple and novel specificities of monoclonal antibodies O1, O4, and R‐mAb used in the analysis of oligodendrocyte development

Abstract

Three monoclonal antibodies that react with antigens on the surface of developing oligodendrocytes in a stage-specific manner, O1, O4 (Sommer and Schachner, 1981), and R-mAb (Ranscht et al., 1982), have been studied with respect to their specificities for a number of purified lipids. The observed specificities were consistent regardless of how the antigens were presented to the antibodies. O1 reacted with galactocerebroside, monogalactosyl-diglyceride, and psychosine and, in addition, labeled an unidentified species in rat brain extracts. R-mAb reacted with galactocerebroside, monogalactosyl-diglyceride, sulfatide, seminolipid, and psychosine; the reaction of R-mAb with sulfatide was nearly equal to that with galactocerebroside. O4 reacted with sulfatide, seminolipid, and to some extent with cholesterol. However, oligodendrocyte progenitor cells labeling with O4 that had not yet begun to express the O1 antigen failed to incorporate 35SO4 or [3H]galactose into sulfatide or seminolipid, the syntheses of which first appear in O1-positive cells. Therefore, O4 stains, in addition to sulfatide and seminolipid, and unidentified antigen that appears on the surface of oligodendrocyte progenitors prior to the expression of sulfatide and galactocerebroside. In primary cultures of rat brain, developing O4+ oligodendrocyte progenitors stained slightly earlier with R-mAb than with O1, and thus R-mAb transiently stained a larger population of oligodendrocytes than did O1. None of the three antibodies produced a detectable reaction on Western immunoblot after separation of brain proteins on reducing gels. In conclusion, the results show that O4, R-mAb, and O1 have multiple overlapping specificities, including previously unrecognized cross-reactions.

A1 Adenosine Receptor Upregulation and Activation Attenuates Neuroinflammation and Demyelination in a Model of Multiple Sclerosis

“…An examiner who was unaware of the slide identity calculated the percent vacuolar change in the white matter of spinal cords, and quantitative analysis was performed as described previously (Linker et al, 2002). Cultured oligodendrocytes (OLs) derived from adult rat brains were stained with an O1 monoclonal antibody that recognizes galactocerebroside (GalC), a marker for mature OLs (Sommer and Schachner, 1981;Bansal et al, 1989). Goat anti-mouse antibody conjugated to Cy-3 (Jackson ImmunoResearch) was used as the secondary antibody to detect labeled OLs.…”

Aβ40 promotes neuronal cell fate in neural progenitor cells

“…The following antibodies were used: rabbit anti-BrdU antibody (Megabase Research Products), mouse anti-NeuN (Chemicon), rabbit anti-S100B (gift from Dr. Linda J Van Eldik), mouse anti-MAP2 (HM-2 clone) and mouse antig-tubulin (both from Sigma), mouse anti-nestin (BD Bioscience), mouse anti-O4 (gift from Dr. Steven Pfeiffer), 38 rabbit anti-Ki67 (ABCam), mouse anti-Ab (4G8 clone, Signet), rabbit anti-activated caspase-3 (Cell Signaling), and Alexa Fluor 594 goat anti-mouse and Alexa Fluor 488 goat anti-rabbit (Molecular Probes).…”

“…For mouse anti-O4 staining, live cells were stained following a protocol described previously. 38 Images were collected with  20 or  40 objectives on a Nikon Eclipse 600 fluorescence microscope. The microscope was equipped with a CoolSnap camera (Photometrics).…”

The localization and non‐genomic function of the membrane‐associated estrogen receptor in oligodendrocytes

“…The O4 hybridoma (Sommer and Schachner, 1981) was kindly provided by Dr. S. Pfeiffer, University of Connecticut. The O4 monoclonal antibody was prepared by following the original protocol (Bansal et al, 1989;Sommer and Schachner, 1981). Reagents used for cell culture were purchased as indicated previously (Boggs et al, 2008).…”