RNA was extracted from the heads of 3 dpf wild type zebrafish and used to perform RT-PCR with degenerate or specific OR primers that amplified ORs belonging to the OR111, OR106, OR128, OR133, OR125, or OR103 subfamilies. PCR products (~ 650 bp long) were cloned into the pCR II-TOPO vector and recovered OR sequences were identified by sequencing. Digoxigenin and fluorescein labeled antisense RNA probes were generated from OR-containing plasmids by in vitro transcription (T7 and Sp6 from Promega Corp.). Embryos were incubated in 0.2% DEPC-Collagenase 25 °C for 2 h, hybridized with OR probes in DEPC-HYB+ and in situ signals were amplified using a Cyanine 3-coupled tyramide signal amplification system (TSA Plus Cyanine 3 System, Perkin Elmer, Product number: NEL744001KT), a Cyanine 5-coupled tyramide system (TSA Plus Cyanine 5 System, Perkin Elmer, Product number NEL745001KT) or a Fluorescein-coupled tyramide system (TSA Plus Fluorescien System, Perkin Elmer, Product number NEL741001KT) as described in Chalasani et al. [33 (link)].
Tsa plus fluorescien system
Manufactured by PerkinElmer
The TSA Plus Fluorescien System is a laboratory equipment designed for fluorescence detection and analysis. It utilizes the technique of Tyramide Signal Amplification (TSA) to enhance the sensitivity of fluorescent labeling. The core function of this system is to amplify and detect low-abundance targets in biological samples.
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Lab products found in correlation
2 protocols using tsa plus fluorescien system
1
Identifying Olfactory Receptor Genes in Zebrafish
2
Dam-fusion Immunofluorescence Localization
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For immunofluorescence studies of the localization of the Dam-fusions, HeLa cells were grown on poly-lysine coated coverslips in a 24-well chamber and co-transfected with the pIND-(V5)-EcoDam-PP1 isoforms or the pIND-(V5)-EcoDam-PIP-WT/M vectors and the pVgRXR plasmid encoding the Ecdysone and Retinoic X receptors (Invitrogen, Waltham, USA). Twenty hours post transfection, 2 μM of Ponasterone A (Invitrogen) was added and 24 h later, the cells were treated for 4 min at 4°C with ice cold CSK buffer (100 mM NaCl, 300 mM sucrose, 3 mM MgCl2, 10 mM PIPES at pH 6.8) supplemented with 0.2% Triton X-100. Subsequently, the cells were fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100, blocked in 3% BSA-PBS and incubated first overnight in 1% BSA-PBS with the primary Dam antibody and then with Horseradish peroxidase (HRP)-conjugated secondary anti-mouse antibody for 2 h. The HRP signal was enhanced by using the TSA-Plus Fluorescien System (PerkinElmer). DNA was stained with DAPI. The cells were visualized with a Leica TCS SPE laser-scanning confocal system mounted on a Leica DMI 4000B microscope, equipped with a Leica ACS APO 40× 1.30NA oil objective.
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