[Histonet] RE: controls and secondaries
gcallis <@t> montana.edu
Wed Apr 13 10:21:40 CDT 2005
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
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
*****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.
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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