Bx dsu

Following a brief rinse in phosphate buffer 0.01 M, mice were perfused with 4% paraformaldehyde and 14% picric acid; brains were post‐fixed for at least 2 hr and then cryoprotected in a 50/50 mixture of fixative and 20% sucrose in 0.01 M phosphate‐buffered saline (PBS) for at least 24 hr. Sections were cut at 60 μm on a sledge microtome with a freezing stage (Yamato REM‐710 electrofreeze MC‐802A), washed in PBS and incubated in 20% normal goat serum. Primary antibodies to tyrosine hydroxylase (rabbit polyclonal 1:5,000, Enzo Life Sciences) or glial fibrillary acid protein (GFAP, rabbit polyclonal, Dako; 1:500) were incubated overnight at 4°C and stained with secondary goat antirabbit antibodies (Life Technologies; 1:200). At least 2 hr were allowed for binding before rinsing in PBS. Sections were mounted on slides; Vectamount AQ (Vector) or occasionally Santa Cruz mountant with DAPI was used to fix the coverslips. A spinning disk confocal microscope (Olympus BX‐DSU) was used and pictures were taken using Neurolucida software and a Hammatsu (EM‐CCD C91) camera.

Images were taken with an Olympus BX61 upright microscope equipped with a spinning disk unit (BX-DSU, Olympus, Tokyo, Japan) and a 10× air objective (UPLFL PH 10×/0.30, Olympus), a 40× water objective (UAPO W/340 40×/1.15, Olympus), or a 60× oil objective (PLAPON O 60×/1.42, Olympus) and an Orca-R² camera (Hamamatsu, Shizuoka, Japan) with Olympus CellSens Dimension 2.2 software. Cilia were visualized with 60× or 40× objectives and images were acquired with 20 planes for 40× images, with a 0.32-µm distance between planes. For 10× and 60× images, 3.54 and 0.24 µm distances were applied, respectively. The number of planes was variable. High magnification images were 2D deconvolved (nearest neighbor) using Olympus CellSens Dimension 2.2 software. Maximum intensity z-projection was created with the same software, and all images were analyzed and equally modified in Fiji/ImageJ [73 (link)].