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Thereafter, the rats were sacrificed, and the lumbar spinal cord and the brain were mounted in Tissue-Tec OTC embedding compound, and deep frozen.
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The brains were removed and post-fixed (24 hr, 4°C) in 4% PFA in PBS, incubated in 30% sucrose (12 hr), and the blocked brain was mounted in cryo-embedding media (OCT, Ted Pella, Redding, CA, United States) on a cryostat for sectioning.
Brains were mounted in Vectashield (Vector Labs) using small cover slips as spacers and analyzed with a LeicaSP5 2photon confocal microscope.
Frozen brains were mounted in a cryostat microtome and 18 µm sections were cut with a knife temperature of -14°C and a sample temperature of -16°C.
Stained larvae brains were mounted in 70% glycerol, 30% Tris pH 7.6 and viewed using bright-field microscopy (Nikon, Eclipse E600).
After further washing, the brains were mounted in Vectashield (Vector Laboratories).
Brains were mounted in VectaShield (Vector Labs, Burlingame, CA).
After washes, brains were mounted in Vectashield Mounting Medium (Vector Laboratories, Inc., Burlingame, VT).
Following 3 × 10 min washes in PBST, brains were mounted in vectashield.
Host embryos and adult brains were mounted in 1.2% agarose/5% sucrose as described above.
Embryos and brains were mounted in VECTASHIELD (Vector) mounting medium and analyzed by confocal microscopy (Leica SP5) using identical settings between controls and mutants (gcm GOF and hypomorph).
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