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Immunofluorescence Staining of Cells

controls & quantify

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#1 moljul

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Posted 07 March 2012 - 02:30 AM

Which controls have to be included in immunofluorescence staining of cells? e.g. Isotype control, "2nd-antibody-only" control...

Is it only a qualitative method, or could quantifications on expression levels (fluorescence intensity) be made?

thx in advance

#2 Rsm

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Posted 07 March 2012 - 04:57 AM

Unstained and 2nd only should do, you can use an isotype control if you want. I always try without controls (most of the time it doesn't work initially, so I don't waste my precious samples), and later use controls. If necessary.

You can do a quantification if you have something next to it in the same picture to compare it to. Like, one cell is transfected and the next one is not. Then you can compare these two cells with imageJ for example. Some people compare stuff on different slides ("parameters were maintained"), but I don't believe in that. I would reject it if I were the reviewer (my old boss used to say that, and I didn't like it. Now I start it myself.).
I got soul, but I'm not a soldier




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