[Histonet] Re: Muscarinic receptor detecting

[J. A. Kiernan]

At 10:50 AM 9/6/03 +0800, Liu Shao Hui wrote:
> ...So far all the antibodies light up the whole cell 
> with no intensified staining on the plasma membrane.

The universal false positives indicate that you are
doing something wrong in every variation of the method.

Do you understand all the steps of the immunohistochemical
methods you are using?  Can you explain the function of
every ingredient of every solution that you apply to the
sections?  How did you determine the dilutions of the
primary and secondary antisera (or antibodies)? 

Your "light up" phrase suggests that you use fluorescence
microscopy. This introduces many other complications, 
notably autofluorescence of the tissue. 

Your controls should determine the step that introduces
the false positive immunostaining. If you are doing 
research (= finding out something new) you should be
using at least 3 control sections for every one that 
might possibly contain a true new result.

Have you learned the principles that determine how
immunohistochemistry is done, or are you following 
instructions that may not be appropriate to your
particular needs?

Nobody should do immunohistochemical staining by
following a "protocol" (horrid misused word) without
knowing the reason for each step of the method. 


John Kiernan
London,  Canada.
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