Anti ncbe

Following postfixation of the brain samples in formalin, 10‐μm thick coronal sections were cut using a cryostat (Leica Microsystems LM3050S). Brain sections revealing choroid plexus were used for immunohistochemical staining as previously described.15 Briefly, these brain sections were incubated overnight at 4°C with the following primary antibodies: anti‐IRP1 (1:200, Abcam), anti‐IRP2 (1:100, Abcam), anti‐NCBE (1:50, Origene), and antitransthyretin (1:50, Abcam). Sections were then washed and incubated with the appropriate secondary antibodies (1:100, Jackson ImmunoResearch Labs) for 2 hours at room temperature. The tissue slides were then stained with 4’,6‐diamidino‐2‐phenylindole (Vector Laboratories), fixed in paramount, and visualized using a fluorescence microscope (Olympus BX51, Olympus Corporation). For histological, volumetric analysis, 16‐μm thick coronal brain sections were cut at 2.5 mm, 1.2 mm, 0.7 mm rostral, and 2.9 mm caudal of the bregma. These brain slices were stained with Nissl and morphometrically analyzed using ImageJ 4.0 (Media Cybernetics) assisted delineation of brain structures, as previously described.16 The ventricle volume was calculated as average ventricular areas on each slide multiplied by the depth of the cerebroventricular system.

Forebrain samples were homogenized in RIPA lysis buffer (Santa Cruz Biotechnology) and supernatants from the homogenates were collected after centrifugation at 20817 g, at 4°C for 30 minutes. The protein concentration was determined using a detergent compatibility assay (Bio‐Rad). Thirty micrograms of protein per sample were loaded, run for 30 minutes at 80 V, then 60 minutes at 120 V, followed by protein transfer onto nitrocellulose membranes at 0.45 mm for 120 minutes (Bio‐Rad). Membranes were incubated for 2 hours in 5% nonfat milk in Tris‐buffered saline containing 0.1% Tween 20. The following primary antibodies were incubated overnight at 4°C: anti‐IRP1 and anti‐IRP2 (1:1000; Abcam), anti‐NCBE (1:500; Origene) and anti–β‐Actin (1:1000; Santa Cruz Biotechnology). The membranes were washed and then incubated with the appropriate secondary antibodies (1:4000, Santa Cruz Biotechnology) for 1 hour at room temperature. Enhanced chemiluminescent solution (GE Healthcare and Life Science) was then applied to the membranes and the protein bands were exposed onto radiography films. ImageJ software (4.0, Media Cybernetics) was used to analyze the relative density of the resultant protein immunoblots.