|Register||Search||Today's Posts||Mark Forums Read|
|Immunohistochemistry Forum Immunohistochemistry Forum. Post and discuss questions on immunohistochemistry methods including paraffin, frozen, or free floating tissue sections.|
| ||LinkBack||Thread Tools||Display Modes|
Multiple antigen immunostaining
It's my first message ... well, i would like to detect 2 antigens simultanously by immunohistochemistry on fixed paraffin-embelled tissu section . So i have chosen ABC method for detection : horseradish peroxydase (AEC) for the first antigen and Alcaline phosphatase (bleu substrat) for the second.
I would like to know if i have taken the right choose and i a little bit confused because the most used chromogen in the article is DAB. AEC is rarely used?! I would like to know the raisons
Re: Multiple antigen immunostaining
Well, in my opinion, fluorescence is the way to go. Because the fluorfores on each antigen of interest will fluoresce at different wavelengths, fluorescent or confocal microscopes will be able to image each antigen staining individually, and if properly done, take the guesswork out of discriminating between your two signals.
If you don't have access to fluorescent scopes, then two color IHC is an alternative (as you suggest).
I would first optimize each antigen individually, and be sure to choose color that are easily distinguishable from each other. for example...don't choose brown and red. Next to each other, they both look brick colored.
However, you may find you need to "retrieve" the antigen if they've been fixed in paraffin. If you google antigen rescue, you'll find many common methods. In our hands heating the slides in sodium citrate buffer works well. Keep in mind the optimal antigen retrieval time may be different for each of your antigens (if it's required at all), in which case you'll have to choose a happy medium between the two times.
Give it a try and let us know how it goes.
|antigen , immunostaining , multiple|
|Thread||Thread Starter||Forum||Replies||Last Post|
|Human Cytome Project - Update 24 Jan. 2005||Peter Van Osta||Cell Biology and Cell Culture||1||08-01-2010 02:18 PM|
|[Drosophila] please help, problems with immunostaining Dros larvabrain||Joan Sim||Drosophila Forum||0||10-03-2009 08:54 AM|
|Antigen Microarray||stanleygeorge||Microarrays Forum||3||06-16-2009 02:41 PM|
|Immunostaining of Condensin complex onto Drosophila chromosomes||Bioelfa||Antibody Forum||0||09-25-2008 06:41 AM|
|Detection, markers and prognostic/predictive significance ofdisseminated tumor email@example.com||Cell Biology and Cell Culture||0||04-10-2008 04:56 AM|