Hello everybody!
I need to perform new experiment. I want to stain collagen fibers in porcine tissue by means of mouse primary antibody. Then to visualize it I am going to use anti-mouse goat secondary antibody Alexa Fluor 488. How should I prepare antibody dilution buffer. Is it ok to prepare 10% normal goat serum (NGS) in PBS blocking buffer and than 10% NGS in PBS for primary mouse antibody dilution buffer and 10% NGS in PBS for goat secondary anti-mouse antibody. Can I dilute goat secondary antibody in goat serum?
Normally, one uses PBS, containing small concentrations of a cheap, non-interfering protein as carrier (say, 0.1% non-fat milk powder or BSA) to prevent unspecific binding of antibodies. In some cases, low concentrations (0.05%) of non-ionic detergent (Tween or Triton) are also added.
The normal serum you use as a negative control: If you incubate your cells with normal mouse serum instead of primary antibody, you should not get any staining if your antibodies are specific. Similarly, you could use normal goat serum instead of secondary antibody, a positive result would indicate that the detection enzyme (peroxydase or phosphatase) is present in your cell.
10% NGS is fine for blocking. You can use 1%NGS for primary and secondary antibody dilution. Diluting goat secondary antibody in goat serum is absolutely fine.
You can add tritonX with PBS in blocking and primary antibody buffer. For detail Islam et al., Neuroscience. 2017 Jan 6;340:201-217. doi: 10.1016/j.neuroscience.2016.10.053.
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