A One-step Immunohistochemical Method for Detection of Blood-brain Barrier Disturbances for Immunoglobulins in Lesioned Rat Brain with Special Reference to False-positive Labelling in Immunohistochemistry

Disturbances of the blood-brain barrier (BBB) following brain lesions lead to extravasation of serum proteins that can be detected by immunohistochemical methods in tissue sections. Here, extravasated immunoglobulins were visualized by a 1-step technique using rabbit anti-rat immunoglobulins conjugated to horseradish peroxidase (HRP). This method is associated with a lower background staining than the conventional 3-step peroxidase-antiperoxidase (PAP) technique using rabbit antibodies against rat whole-serum proteins or immunoglobulins (IgG). Further tests using a direct conjugate of rabbit anti-rat immunoglobulins to fluorescein isothiocyanate (FITC) showed usefulness of the approach for fluorescence microscopy. Additional experiments showed that antibodies directed against mouse immunoglobulins as used for detection of mouse monoclonal antibodies can cross-react with extravasated rat immunoglobulins. Therefore, immunohistochemical studies on lesioned rat brain should routinely include a visualization of areas containing extravasated serum proteins including immunoglobulins.