[Histonet] RE: controls and secondaries

From:Gayle Callis

What is the difference between  a secondary such as a goat anti- mouse
and a straight mouse IgG istotype control as far as it's applications in
the IHC process?

*****The IgG isotype is used as the negative control, and must match the 
concentration of your primary.  This is preferred to using normal mouse 
serum as a negative control since serums can contain nonspecific antibodies 
which can cause nonspecific background.  I used normal serum one time and 
publication reviewers balked big time, I had to go back and redo ( a VERY 
difficult experiment) ALL my immunostaining using a mouse IgG isotype 
control. Haven't use normal serums of any sort since that time.

So to set up your IFA or even IHC staining, you would have a primary (mouse 
anti-whatever) on one section and mouse IgG (it may be IgG1, IgG2a, etc, 
etc) at same concentration your primary on a separate section labeled 
negative control.

at tI ask because someone from another lab recently came to
me for help with a long list of stains they need done in a short period
of time. They have already ordered everything they think they need to
complete the stains. Most of them are flouescent conjugated primaries,
but two will have to have the fluorescence attached to the secondary.

*****Extra comment here: If the directly conjugated primary mouse 
antiwhatever-FITC fails to work, you can use DAKO's rabbit antiFITC, then 
come back with goat antirabbbit-FITC OR Donkey antiRAbbit-FITC.  I have had 
primaries that work in FACS that will NOT work i.e. fluoresce on tissue 
sections.  It is a spatial, stoichiometry (sp?) problem, but easily solved 
in a rather simple way, an extra two steps. Do not buy DAKO's antirabbit 
conjugated to anything, it doesn't work as well as I described.  I hope 
this does not happen to you.

**** IF your primary is conjugated to FITC or any other fluorophore, then 
the mouse IgG isotype must be conjugated to FITC also in order to have a 
correct negative control.  If you have a problem with this, you can get 
mouse IgG (which contain ALL the isotypes in one whole IgG) conjugated to 
FITC from Jackson (fast, cheap and excellent) - still legal to use as long 
as the isotype is there.

I am wondering about this because I was asked why their protocol would
call specifically for goat serum to block, and would it matter if it was
another species.

*****As long as they are using goat antimouse, then the blocking serum is 
goat.  This is what we do here. Blocking serum, even though you are using a 
directly FITC conjugated mouse antiwhatever could be goat, that is not 
uncommon. Swine, in your case would be avoided, but some use donkey, horse 
but we prefer to do what you said in next statement.

I told them that usually the blocking serum is the same
as the species the secondary was made in and was told that the secondary
wasn't made in anything different such as a goat anti-mouse, they had
what I would normally use as control serum.

???Question here:  are you saying your control serum is mouse serum and 
used as the negative control?  As said in beginnign, can contain 
nonspecific antibodies that could cause background as I said in beginning.

*****If you are using a goat antimouse, the blocking serum should be goat 
serum.  It can be used as or in the diluent for the goat antimouse-FITC 
secondary also.  With FITC conjugated secondaries, we would use approx 2 - 
5% goat serum in diluent and no BSA. If you were using donkey antimouse, 
then the blocking serum becomes donkey serum.  A word to the wise when 
working with large animals, it is a good idea to have purchased a seondary 
adsorbed to the species being stained.  In your case that would be Goat 
antiMouse-FITC adsorbed to swine.

Can it be used as a  secondary? I have never tried this. Supposedly it 
worked with their flow
samples as a secondary (these are mouse primaries on pig tissue).

****I got lost here, you ARE using a goat antimouse (conjugated to FITC?)to 
detect your mouse antiwhatever on pig tissue section, correct?  And that is 
what they used in FACS analysis too?

If you wish, I will be happy to discuss this further privately.

Gayle Callis
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-6367 (lab with voice mail)
406 994-4303 (FAX)

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