Immunohistochemical and Immunocytochemical Detection of Phosphoproteins in Striatal Neurons - Drugs of Abuse: Neurological Reviews and Protocols - page 275

dilution from the original solution of commercial antibody regardless of its real
concentration. In this case, 1 1000 dilution from a 0.1 mg/mL antibody package

actually means 1 10,000 dilution (0.1 µ g/mL). Checking with the original author

could help verify this issue.

4. Variation in immunostaining density exists among the procedures performed at
different times even though the identical procedures are followed. It is therefore
highly recommended to conduct the detection of a given protein simultaneously
on the sections or culture slides from different experimental groups. By perform-
ing the same procedures at the same time, the density variation is expected to
be minimal. Changes in the immunostaining density can therefore be compared
quantitatively among groups of animals treated with different drugs.

5. The secondary species serum (normal goat serum) incubation allows for the
binding of serum protein to charged sites on the brain section. The concentration
of the normal goat serum and the duration of incubation may be adjusted to
reduce nonspecifi c background staining.

6. Primary antibodies should not be thawed and refrozen frequently. They should
be stored in aliquots with or without dilution of the original package and thawed
once and used. However, most antibodies used in this study can be saved in
solution at -20°C, which avoids freeze-thaw cycles during their frequent uses.

7. Some frozen tissues, most commonly kidney and liver, show intrinsic biotin-
binding activity, which may increase the nonspecific background staining.
However, such a problem is not signifi cant when striatal sections are proceeded
for immunohistochemistry. By using the Blocking Kit from Vector to block the
intrinsic biotin-binding activity (adding avidin from the kit to the brain section
to bind to biotin, followed by biotin to bind avidin and rinse), we observed
a high signal-to-noise ratio comparable to that obtained without the blocking
treatment.

8. We add nickel chloride to the DAB solution to increase the overall sensitivity
of the DAB reaction. The DAB reaction product after nickel chloride addition is
dark blue to black, instead of brown produced by the utilization of DAB alone.
The dark reaction product seems preferable for black and white photography
of stained slides.

9. If the counterstaining is desired, this stain is better remained weak. Strong
counterstaining can markedly mask the principal reaction product. Usually,
the counterstaining is controlled to intensity suffi cient just to visualize the
structure.

Acknowledgments
This work was supported by NIH Grants DA10355 and MH61469.

References
1. Churn, S. B. (1995) Multifunctional calcium and calmodulin-dependent kinase II
in neuronal function and disease. Adv. Neuroimmunol. 5, 241-259.