Brain–ring gland complex was dissected out in cold phosphate-buffered saline (PBS) buffer and fixed in 4% paraformaldehyde for 20 min (90 (link)). Brain–ring gland complex was stained overnight at 4°C with chicken anti-GFP (1/500; Life Technologies), and rat anti-Pvr (91 (link)) (1/500; a gift from B. Shilo). Secondary antibodies 488 anti-chicken (1/500) and 647 anti-rat (1/500) were purchased from Invitrogen. For DNA and nuclei staining, we used Hoechst (1/1000) for 10 min at room temperature. We set up the tile scan with the micrometer-thick Z stack on an LSM880 Zeiss microscope with 40× oil objective. Images were acquired with a resolution of 1024 × 1024.
488 anti chicken
Manufactured by Thermo Fisher Scientific
The 488 anti-chicken is a fluorescently labeled antibody used for the detection and identification of chicken-derived proteins or antigens in research applications. It emits fluorescence at 488 nanometers when excited, allowing for the visualization and analysis of target proteins or cells.
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2 protocols using 488 anti chicken
1
Brain-Ring Gland Imaging Protocol
2
Whole-mount and Cryosection Immunolabeling
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Whole-mount immunolabeling procedures were performed as previously described (Wilson et al., 1990 (link)). For antibody staining of cryosections, embryos were first protected by sequential incubation in 15% then 30% sucrose in phosphate-buffered saline supplemented with 0.5% Triton X-100 (PBST) for 30′, embedded in OCT, stored at −80°C, and sectioned at 18 μm using a Leica cryostat. Primary antibodies were as follows: mouse anti-zonula occludens 1 (ZO1; 1:600, Sigma), rabbit anti-laminin (1:600, Sigma), chicken anti-GFP (1:1,000; Sigma). The secondary antibodies were: Alexa Fluor 633 anti-mouse, 488 anti-rabbit, and 488 anti-chicken (all 1:1,000, Invitrogen). Images were collected on a Leica confocal microscope using a 40x oil immersion lens. Gain and offset were adjusted to enhance the contrast of the signal against the background.
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