PA-GFP positive larvae were raised in a dark incubator to prevent background photoconversion. Larvae were anesthetized with 0.2 mg/mL ethyl-3-aminobenzoic acid ethyl ester (MESAB, Sigma-Aldrich E10521, St. Louis, MO) and mounted laterally in 2% low-melting temperature agarose (Thermo Fisher Scientific 16520). Using a Zeiss LSM800 confocal microscope with 20x objective (Zeiss W Plan-Apochromat 20x/1.0 DIC CG=0.17 M27 75mm), the spinal cord between the mid-point of the swim bladder and the caudal-most tip of the tail was repeatedly scanned with a 405 nm laser until fully converted. For retrograde labelling of vestibulospinal neurons used for photoablations, the spinal cords of Tg(α-tubulin:C3PA-GFP) larvae were converted at 6 dpf. To allow the converted fluorophore to diffuse into neuron bodies, all fish were removed from agarose after photoconversion and raised in E3 in a dark incubator.
W plan apochromat 20x 1.0 dic cg 0 17 m27 75mm
Manufactured by Zeiss
The W Plan-Apochromat 20x/1.0 DIC CG=0.17 M27 75mm is a high-performance objective lens designed for optical microscopy. It features a magnification of 20x and a numerical aperture of 1.0, providing excellent image resolution and contrast. The lens is optimized for use with a cover glass thickness of 0.17 mm and has a thread mount of M27.
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2 protocols using w plan apochromat 20x 1.0 dic cg 0 17 m27 75mm
1
Photoconversion of Spinal Neurons in Larval Zebrafish
2
Immunohistochemical Staining of Brain Tissue
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Brains were dissected in PBS and then fixed for 14 minutes in 4% paraformaldehyde in PBS. After fixing, brains were washed 3 times in PBS and stored at 4°C until staining (≤1 week). Brains were incubated in 5% normal goat serum in PBST (0.2% Triton-X in 1x PBS) for a minimum of 60 minutes and then incubated overnight in primary antibody. Brains were then washed 3x with PBST and incubated overnight in secondary antibody. Brains were washed 3x in PBST, then mounted, covered in vectasheild (Vector Labs H-1000) and enclosed with a coverslip prior to imaging with a 20x objective (Zeiss W Plan-Apochromat 20x/1.0 DIC CG 0.17 M27 75mm) on a Zeiss LSM 800 confocal microscope at 1.25 μm depth resolution. All images were created in Image J using a maximum z-projection. Antibodies used were as follows:
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