Dave - you are right - the negative controls for ImmunoStains can become
quite complex (isotypes, concentrations, animal species, absorbed specific
antibodies .....)

We just had our latest CAP inspection a few weeks ago.  In the disk version
the following commentary is stated which  should be very interesting to use
Histonetters.
### CHECKLIST : SECTION 08 : VERSION 19981 ###NOTE: Checklist questions,
including those with explanatory text,are NOT Standards. They are tools for
inspectors and directors to use in evaluating whether or not the laboratory
is meeting the Standards for Laboratory Accreditation.

 "QUESTION: 08:2257 PHASE: II NEW Are negative controls used for each
antibody? YES COMMENTARY: 08:2257 PHASE: II NEW    NEGATIVE CONTROLS MUST BE
INCLUDED FOR EACH TISSUE EVALUATED. ISOTYPE-SPECIFIC CONTROLS ARE
ACCEPTABLE, BUT NOT REQUIRED. NON-IMMUNE IMMUNOGLOBULIN FRACTIONS
EQUIVALENTLY DILUTED TO PRIMARY ANTIBODY CONCENTRATIONS ARE PREFERRED. IF
PRIMARY ANTIBODIES FROM MORE THAN ONE SPECIES ARE USED, NON-IMMUNE
IMMUNOGLOBULIN NEGATIVE CONTROL SHOULD BE EMPLOYED FOR EACH REAGENT SPECIES.
BUFFER SUBSTITUTION CONTROLS ARE ACCEPTABLE IF AT LEAST THREE PRIMARY
ANTIBODY REAGENTS FROM THE SAME SPECIES, ONE OF WHICH IS NEGATIVE FOR THE
TARGET ANTIGEN ARE UTILIZED, THEREBY SERVING AS A DE FACTO IRRELEVANT,
ANTIBODY CONTROL. "

At our lab we run neg. controls in the following manner :
1. For each type of  primary specific antibody  (mouse, rabbit, etc), we use
a species-specific, non-immunized host animal serum to look for non-specific
staining .  This reagent is applied on one of  the positive (and internal
negative staining tissue) tissue controls and the patient sections.
2.  I  like to use the negative control serum purchased from the vendor of
the specific antibodies used to stain the patient sections.

I know that the use of these negative control reagents satisfies the
inspectors.  I  have utilized isotype-specific (IgG1, IgG2, etc)  negative
controls in immunophenotyping leukocyte populations by flowcytometric
methods, I have never used these in Immunohistology.  Has anyone else?

I have always utilized the staining (or lack of staining) results of all
antibodies used as primary antibodies to help interpret minimal or
background staining.  I do not recommend the use of a buffer control.

Mark Briones
Valley Children's Hospital
Madera CA   USA