Publication protocol
Immunohistochemistry was conducted as previously described by Olmsted-Davis et al.20 Primary antibodies were used at a dilution of 1:100–1:200 and secondary antibodies (Alexa Fluor 488, 594, or 647; Invitrogen Life Technologies, Carlsbad, CA) at a 1:500 dilution. Primary antibodies used were as follows: rabbit polyclonal antibodies (ADBR3) (Abcam, Cambridge, UK), Ki67 (Abcam, Cambridge, UK), early growth response protein 2 (EGR2) (Proteintech Group, Inc., Rosemont, IL) and mouse monoclonal antibodies beta 3 tubulin (TUJ1) (Promega, Madison, WI), and neurofilament 1 (NF1) (Sigma-Aldrich, St. Louis, MO). Primary and secondary antibodies were diluted in PBS with 2% bovine serum albumin. Tissues were counterstained and covered with Vectashield mounting medium containing 4′,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Burlingame, CA). Stained tissue sections were examined using an Olympus BX41 microscope (Olympus Corporation of the Americas, Waltham, MA) equipped with a reflected fluorescence system, using a 10×/0.40 numerical aperture objective lens. Stained tissue sections shown in Figure 1(d) were examined by confocal microscopy (Zeiss Inc, Thornwood, NY, LSM 780). To ensure signal specificity, controls were performed, and the specific absorption spectrum from each primary–secondary pair was captured. Lambda mode was then used to separate signal into each component. In this way, autofluorescence was filtered out.
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