From Marrow to Brain: Expression of Neuronal Phenotypes in Adult Mice
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Tài liệu tham khảo
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Gurdon J. B., J. Embryol. Exp. Morphol. 10, 622 (1962).
Marrow was isolated in a sterile environment from 8- to 10-week-old male transgenic mice that ubiquitously expressed enhanced green fluorescent protein (GFP) and non-GFP control mice. After lethal irradiation 8- to 10-week-old C57BL/6 mice (Stanford) received 6 × 10 6 cells by tail vein injection.
Isolated brains were minced with a razor blade dissociated with proteases washed stained with TriChrome (TC)–conjugated rat antibody to mouse CD11b and allophycocyanin (APC)–conjugated rat antibodies to mouse CD45 and analyzed by flow cytometry.
Each GFP + cell was analyzed for antibody staining by three-dimensional confocal laser scanning microscopy. Data was collected with sequential laser excitation to eliminate bleedthrough and with confocal parameters (e.g. pinhole sizes) selected to minimize the thickness of the calculated optical section.
Eight or 12 weeks after bone marrow transplant experimental mice and age-matched control mice were perfused and fixed with 1.5% paraformaladehyde (PF)/0.1% glutaraldehyde snap frozen in TISSUE-TEK O.C.T. compound cryosectioned and stained as floating sections with antibodies against NeuN 200-kD neurofilament class III β-tubulin GFAP F4/80 and CD45. All sections were blocked with 25% normal goat serum 0.25% Triton-X 100 and antibody to CD16/32. Goat antibodies to mouse and to rabbit conjugated to Texas Red or Cy5 were used as secondary antibodies.
Mice were anesthetized with Methoxyflurane and surgically decapitated and the OBs were rapidly isolated and incubated in Tyrode solution. OBs were fixed in 1.5% paraformaldehyde/4 mM EGTA and were sliced to yield coronal sections. The samples were blocked and permeabilized in 0.3% Triton-X 100 3% bovine serum albumin (BSA) monoclonal antibody to mouse CD16/CD32 and 100 mM glycine. Staining was performed with polyclonal antibody to pCREB and monoclonal antibody to NeuN washed and stained with secondary antibodies (Texas Red goat antibody to rabbit; Cy5 goat antibody to mouse).
Web video and supplemental text are available at Science Online at www.sciencemag.org/cgi/content/full/290/5497/1775/DC1.
We wish to thank neurobiologists S. McConnell K. Deisseroth and J. Weimann for their expertise and ongoing guidance; U. Wang and S. Heck for technical expertise; B. Blakely for insightful comments; M. Okabe for transgenic GFP mice; and M. Greenberg for antibody to pCREB. This research was supported by the Life and Health Insurance Medical Research Fund and a NIH predoctoral training grant (T.R.B.) a fellowship from Human Frontiers in Science Program (F.M.V.R.) a postdoctoral fellowship (G.I.K.) and NIH research grants CA59717 AG09521 and HD18179 (H.M.B).