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Dear all,

Sorry this is so long -

Use permeabilized cells and substitute primary host immunoglobulin at the
same working concentration as  primary antibody. Example: if primary is
mouse anti "subunits of the Na/K-ATPase", mouse IgG2a indicating
immunoglobulin subclass of mouse host, then the negative control is Mouse
IgG2a. Whole mouse IgG can be used since it is a mixture of all subclasses
or isotypes. This will test if host immunglobulin portion causes
nonspecific staining and this will not be tested IF you do just a PBS
control.  Some people will use primary antibody of irrelevant specificity
in place of whole or isotype matched Ig's.

Caveat: We did not do a proper isotype matched control once, and reviewers
asked for repeat of immunostaining - a harsh lesson!

PBS or diluent subtituted for a primary antibody is fine for checking
nonspecific binding of secondary antibody to cells. We refer to PBS as a
null control. I really liked "no antibodies for checking autofluorescence"
- good point!

To borrow from Francesca's tidy setup a bit, we would set up staining
protocol:
Permeabilized cells with:

1.      Mouse IgG negative
        Goat antimouse F(ab') frag of whole IgG, adsorbed to rat

2.      Mouse anti A
        Goat antimouse (F(ab')2 frag of whole IgG, adsorbed to rat

3.      Rabbit IgG negative control
        Donkey anti Rabbit F(ab')2 fragment of IgG, adsorbed to rat

4.      Rabbit anti B
        Donkey anti Rabbit F(ab')2 fragment of IgG, adsorbed to rat

We use F(ab')2 fragments of IgG to prevent cross reaction to fc receptors
on cells, particularly important in closely related species - rat and mouse

In place of "set up one slide incubating with anti-A (mo)+anti-rabbit
>and another one for anti-B (rb)+anti-mouse.", you can substitute Anti A
antibody with mouse IgG, and rabbit IgG for anti-B, to test if secondary
antibodies are cross reacting to primary antibody host immunoglobulins.

It is a good idea for planned double staining, to do single staining first
to determine nonspecific binding of Mouse IgG with its secondary and same
for rabbit IgG with its secondary, make necessary adjustments, then combine
for double staining.

Gayle Callis
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University
Bozeman MT 59717-3610
email: [log in to unmask]


>Hi Fredrik,
>
>for the first time I would set up the following controls:
>-no antibodies (for autofluorescence)
>-no primary antibodies, incubate only with secondary abs (to check whether
>the secondary abs bind aspecifically your sample)
>-I would also verify that the secondary ab does not aspecifically bind the
>other primary:
>i.e. if you have
>anti-A (raised in mouse)
>anti-B (raised in rabbit)
>anti-mouse
>anti-rabbit
>
>set up one slide incubating with anti-A (mo)+anti-rabbit
>and another one for anti-B (rb)+anti-mouse.
>
>Good luck,
>Francesca
>

>>From: Fredrik Swift <[log in to unmask]>
>>Date: Monday, May 12, 2003 1:52 pm
>>Subject: Controls in immunolabelled cells
>> > Hello,
>> >
>> > I'm planning some immunolabelling experiments. I will use primary
>> > antibodiesagains subunits of the Na/K-ATPase and the Na/Ca-
>> > exchanger in permeabilized
>> > rat cardiomyocytes and secondary antibodies coupled to an Alexa-
>> > fluorochrome(Mol. Probes). What would be appropriate controls in
>> > this type of
>> > experiment?
>> > - Without primary antibody?
>> > - Non permeabilized cells?
>> > - Any other good positive/negative controls?
>> >
>> > Thanks,
>> >
>> > Fredrik

Gayle Callis
MT,HT,HTL(ASCP)
Research Histopathology Supervisor
Veterinary Molecular Biology - Marsh Lab
Montana State University - Bozeman
S. 19th and Lincoln St
Bozeman MT 59717-3610

406 994-6367 (lab with voice mail)
406 994-4303 (FAX)

email: [log in to unmask]