2: Data Acquisition

Magnetic resonance imaging is done at 37° C using an 89 mm vertical bore 11.7 T Bruker Avance imaging spectrometer with a micro-imaging gradient insert and 30 mm birdcage RF coil (Bruker Instruments). Typical imaging parameters are as follows: T2-weighted RARE 3D imaging protocol (8 echoes), matrix dimensions = 256 x 256 x 256; FOV = 3 cm x 1.5 cm x 1.5; repetition time (TR) = 1500 ms; effective time (TE) = 10 ms; number of averages = 4. The images are padded with zeros to double the number of time domain points in each dimension, the Fourier transform yields a matrix of 512 x 256 x 256. This procedure is commonly called “zero-filling” and is a well known interpolation method. Typical spatial resolution is approximately 60 µm3 per voxel.

Blockface Imaging
The brains are attached to a chuck with OCT compound (Sakura), and sections are cut serially in 50µm thick coronal sections on a modified CM3050S cryostat (Leica). A DMCIe digital camera (Polaroid) captures images of the blockface prior to each section at a resolution of 1600 x 1200 (approximately 6.7µm/pixel) in 24-bit color.

Sections 200µm apart are Nissl-stained (thionin) and alternating sections 200µm apart were myelin-stained using a modified myelin impregnation stain. Immunohistochemistry. Sections 200µm apart were stained with antibodies to myelin basic protein (MBP) (link to protocol), glial fibrillary acidic protein (GFAP), estrogen receptor beta (ERB), and other proteins. Stains were visualized using horseradish peroxidase (HRP) with diaminobenzidine (DAB) as a chromogen.

Stained preparations were digitized using a 1.25X objective on an AX70 microscope (Olympus) with a DMX-1200 digital camera (Nikon) at a resolution of 3840 x 3072 (approximately 3 µm/pixel) in 24-bit color. The images were acquired using a macro imaging system that provided undistorted high-resolution images with even illumination across the entire field of view.