Hello,

Since I'd like to detect a protein existing at very low levels, I'm thinking
to try triple step staining: using a primary Ab, a secondary Ab, then a
fluorochrome conjugated tertiary Ab.  No one around me have done it and I do
not have any idea.  Any suggestions will be greatly appreciated regarding to
especially the following questions...

(1) The protein is intracellular (ER and Golgi).  I wonder whether the
bulkiness of the whole molecules affects staining efficiency...?

(2) Do you know any reference which helps for the triple staining?
Anything, papers/protocols/books, will be welcome.

(3) Or are there any good alternatives, rather than doing the triple
staining, to detect low level proteins?

Thank you very much in advance.

Mari