Dear colleagues,
We want to order some primary antibodies conjugated with alexa fluor and we are confused.
What application of pAB (primary antibodies) with fluorochrome? Can we perform IHC for frozen or paraffin section? Could Alexa Fluor (AF 405 for example) bleach before visualization is made? While washing after incubation?
We are study dopamine receptors, such as D1 and D2. https://www.scbt.com/p/d2dr-antibody-b-10 - this one for DRD2 (AF 647 is available), https://www.novusbio.com/products/dopamine-d1r-drd1-antibody-sg2-d1a_nb110-60017af405 - anti-dophamine receptor 1 with AF 405. The reason of using conjugated antibodies is performed triple (or dual) staining procedure with fluorescent intensity measuring.
Your experience are welcome!
Hallo Svetlana,
your labelled primary ABs do not bleach when the immuno reaction takes place in the dark. You can use a pape-box therefore. Before you start, make a test with different dilution of your primary ABs to find out which is best one for you. If the result is not sufficient you can try byotinylated secondary AB and with fluorphore labelled Streptavidine. This is a more sensitive system. Weather your ABs working on frozen and/or paraffin sections , this you could find out with such a dilution row. You will have a better garantee that your ABs are suitable when you buy ABs where the paticular application which you need is already mensioned.
Good luck!
It's a problem of sensitivity of you immunostaining. if your target is very expressed in your tissu (ex: insulin in pancreas) you can use a primary antibody directly conjugated with a fluorochrome. If not, you can amplify the signal using a primary antibody against you target and a second antibody conjugated against tht first one. Because several secondary antibodies will link the first, you will obtain a amplification of the signal.
Thank you so much Ute Neubacher and Anthony Lucas ! Your answers are very helpful!
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