Perfusion-fixation of the human brain for immunohistochemistry: comparison with immersion-fixation

A method of perfusion-fixation of the human brain is described and compared with immersion-fixation by immunoperoxidase staining for several substances (tyrosine hydroxylase, substance P, choline acetyltransferase, glutamate decarboxylase, Met-enkephalin, and neuron-specific enolase) in human striatum. Results from 1-cm slices fixed by immersion for 1, 2, 4 and 8 days were compared with results from slices of perfused brain postfixed for the same time periods. The fixative used in all steps was 4% paraformaldehyde at 4 degrees C. In the immersion-fixed brains, optimal immunoreaction for tyrosine hydroxylase and glutamate decarboxylase was limited to a depth of 1-2 mm from the surface of the brain slice. In contrast, staining density in perfusion-fixed brains was relatively homogeneous and of high quality. The other antigens studied displayed more uniform staining throughout the section with both perfused and immersed brains. Investigators intending to study human brain immunohistochemistry using immersion-fixation should be aware of the possibility of depth-related variations in staining intensity and would be wise to determine whether this effect is significant for the antigens they choose to study.