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Local Z Projector example dataset and parameters 03

Laure Mancini; Nicolas Dray; Laure Bally-Cuif; Jean-Yves Tinevez

Adult zebrafish brain, imaged on a laser-scanning confocal microscope.

Brains were dissected in 1X solution of phosphate buffered saline (PBS - Fisher Bioreagents) and directly transferred to a 4% paraformaldehyde solution in PBS for fixation. They were fixed for 2 to 4 hours at room temperature (RT) under permanent agitation. After four washing steps in PBS, brains were dehydrated through 5 minutes series of 25%, 50% and 75% methanol diluted in 0.1% tween-20 (Sigma Life Science – P9416) PBS solution and kept in 100% methanol (Sigma-Aldrich, 322415) at -20°C. The whole-mount immunohistochemistry (IHC) started by the rehydration of the telencephali. Then, the brains were subjected to an antigen retrieval step using Histo-VT One (Nacalai Tesque) for an hour at 65°C. Brains were rinsed in a 0.1% DMSO and 0.1% Triton X-100 (Sigma Life Science– 1002135493) PBS 1X solution (PBT) and then blocked with 4% normal goat serum in PBT (blocking buffer) 4 hours at RT. The blocking buffer was later replaced by the primary antibodies solution, and the brains were kept overnight at 4°C on a rocking platform. The next day, brains were rinsed over 24 hours at room temperature with PBT and incubated in a solution of secondary antibodies diluted in PBT overnight, in the dark, and at 4°C on a rocking platform. After several washes, the telencephali were mounted in PBS on slides using a 0.7 mm-thick holder. The primary antibody anti-ZO1 was used at 1:200 (Mouse monoclonal IgG1 anti-ZO1, Thermo Fisher, cat. #33-9100, RRID: AB_2533147) and the secondary antibody anti IgG was used at 1:1000 (Goat anti-Mouse IgG (H+L) Alexa633 conjugated, Thermo Fisher, cat. #A-21052, RRID : AB_2535719).

Images of whole-mounted immunostained telencephali were acquired on confocal microscope (LSM700, Carl Zeiss A.G), using a 40X oil objective. We acquired images with a z-step of 0.65 µm. We averaged each line four times with an image resolution of 1024 ×1024 pixels with a bit-depth of 12-bits. We recorded mosaics with a 15% overlap to image an entire hemisphere per fish.

See the accompanying paper: DProj: A toolbox for local 2D projection and accurate morphometrics of large 3D microscopy images.

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