For double color in situ hybridization and immunostaining, papp-aa mRNA signal was detected using an anti-DIG-POD antibody (Roche), followed by Alexa 488 Tyramide Signal Amplification (Invitrogen). After in situ hybridization analysis, the stained larvae were washed in 1xPBST then incubated with a GFP antibody overnight at 4°C. Larvae were then stained with a Cy3 conjugated Goat anti-Rabbit IgG antibody (Jackson ImmunoResearch, West Grove, PA). Images were acquired using a Nikon Eclipse E600 Fluorescence Microscope with PMCapture Pro six software.
Alexa 488 tyramide signal amplification
Alexa Fluor 488 Tyramide Signal Amplification is a reagent used in fluorescence-based detection techniques. It provides signal amplification, enhancing the detection of targeted biomolecules. The product functions by depositing multiple fluorescent Alexa Fluor 488 labels at the site of the target, resulting in an amplified signal.
2 protocols using alexa 488 tyramide signal amplification
In Situ Analysis of Papp-aa Expression
For double color in situ hybridization and immunostaining, papp-aa mRNA signal was detected using an anti-DIG-POD antibody (Roche), followed by Alexa 488 Tyramide Signal Amplification (Invitrogen). After in situ hybridization analysis, the stained larvae were washed in 1xPBST then incubated with a GFP antibody overnight at 4°C. Larvae were then stained with a Cy3 conjugated Goat anti-Rabbit IgG antibody (Jackson ImmunoResearch, West Grove, PA). Images were acquired using a Nikon Eclipse E600 Fluorescence Microscope with PMCapture Pro six software.
Whole Mount Immunostaining and In Situ Hybridization in Zebrafish
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