I am working on some immunohistochemistry staining of paraffin embedded tissues and am curious to know if there are any guidelines in choosing the species of the normal serum used for a blocking buffer? I understand the host species should match that of the secondary antibody (and should definitely NOT match the species of the primary antibody), but does it matter which species to use in general? Like does using donkey serum give any advantage over using goat or rat serum?
I do not think the blocking serum should match the secondary antibody.
Intuitivly I would not use rat (to close evolutionnary from mouse, where quite a lot secondary antibodies are done).
I use goat serum. works fine
Hi Frances,
As a first try we always use the serum of the species of the secondary antibodie. If that doen't work, it becomes a case of trial and error.
You can also try:
1% BSA in PBS
5% BSA in PBS
1-5% non fat dry mil in PBS
Human AB serum is also an option.
with kind regards,
Ronald
Hi Frances,
From learning perspective, we do consider that blocking buffer must not match with host of the secondary antibody. However once you have enough exposure in IHC, this limitation actually becomes meaningless.
I have used 10% FBS in pbs as a blocking buffer (as well as primary Ab diluent) at RT for 2hrs in paraffin embedded sections. I have always been comfortable with above blocking buffer in case primary host are rat, rabbit or mouse and I have never met with any problem (background) regardless of secondary Ab hosts. Hope it helps.
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