Floating sections were rinsed 3 × 5 min in PBS and incubated for 1 h at room temperature in PBS containing 1% bovine serum albumin and 0.1% Triton-X-100, followed by 72 h of incubation with primary antibody. For BrdU, the formamide treatment described above was also applied. The following primary antibody dilutions were used: 1:1000 mouse anti-BrdU (#AB6326, Abcam, Cambridge, UK), 1:500 mouse anti-NeuN (#MAB377, Millipore), 1:300 rabbit anti-cleaved caspase-3 (#2305-PC, Trevigen,Gaithersburg, MD, USA), 1:2000 anti-GFP (#A6455, Thermofisher, Life Technologies, Carlsbad, CA, USA) and 1:500 goat polyclonal anti-DCX (#8066, Santa Cruz, Dallas, TX, USA). Detection was with Alexa dye-conjugated antibodies (Molecular Probes, Eugene, OR, USA, Thermofisher, Waltham, MA, USA) at a concentration of 1:500. Nuclei were visualized with Hoechst-33342 (1:1000). Tiled images were acquired with a LSM-780 confocal microscope (Carl Zeiss, Oberkochen, Germany) using 10 × , 20 × and 40 × objectives. Images shown are typically from maximum projections of 5 × z-planes.
Alexa dye conjugated antibodies
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alexa dye-conjugated antibodies are fluorescent-labeled antibodies designed for use in various research and diagnostic applications. These antibodies are conjugated with Alexa fluorescent dyes, which offer bright and stable fluorescence signals. They can be used for techniques such as flow cytometry, immunofluorescence, and other imaging applications to detect and visualize target proteins or cells.
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2 protocols using alexa dye conjugated antibodies
1
Immunofluorescence Labeling and Microscopy
2
In Situ Hybridization and Immunofluorescence Staining in Zebrafish
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ISH and immunofluorescence staining were performed according to standard procedures (Lecaudey et al., 2004 (link)). amotl2a, taz, and yap1 ISH probes were amplified by PCR from zebrafish embryo cDNA using the primers indicated in the Supplementary file 1A . PCR products were cloned into the pGEM-T vector (Promega GmbH, Germany) according to manufacturer's instructions. The probes for cxcr7b, cxcr4b, lef1, and prox1 were previously published (Glasgow and Tomarev, 1998 (link); Dorsky et al., 1999 (link); Thisse and Thisse, 2005 (link); Valentin et al., 2007 (link)). The following antibodies were used: rabbit anti-GFP (1:500; Torrey Pines Biolabs, Secaucus, NJ, United States), mouse anti-GFP (1:500; JL8, Takara Bio Europe/Clontech, France), mouse anti-ZO1 (1:500; GmbH), mouse anti-HCS-1 (1:20, HCS-1 was deposited to the DSHB, Iowa city, IA, United States by Corwin, J), and Alexa dye-conjugated antibodies (1:500; Molecular Probes). Rhodamine-phalloidin (Life Technologies GmbH) was used at 1:100.
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