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immunocitochemistry protocol -newbie needs help

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Old 11-16-2006, 08:23 PM
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Default immunocitochemistry protocol -newbie needs help

Hello
I'm a humble graduate student who was given the task to perform immunocitochemistry for the first time in his life.
Protocols were not hard to come by, but I do have a few questions.
The antibody of choice is the biotinylated anti-human integrin alpha2 (CD49b) Ab, from RnD systems (#BAM1233). Since it already has a biotin tag, do I need a secondary antibody at all? (The reference article in the product sheet requires subscription to the Journal of Pathology -which our university does not have. I ordered the article through ILLIAD, but it could take weeks to arrive.)
How does a direct ICC staining protocol look like? (now, those I couldn't find). Everybody uses the indirect method. My guess is that the all the steps between the primary Ab and streptavidin are missing, but I'd like to be sure.
If I am to use a secondary Ab (the lab coordinator did order one), what is the biotin tag good for?
What kind of controls should I use? I'd like to profile the a2b1 integrin-content of different cell lines...
Obviously the method does not allow quantitation. Although the product sheet does not mention it, would whole-cell ELISA be possible instead? (What makes an antibody better for ELISA?) My head is buzzing with questions...
Thank you for taking the time to read my post and any help is greatly appreciated.
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