Modified Beilschowsky silver impregnation combined with Hematoxylin or Cresyl Violet counterstaining as a potential tool for the simultaneous study of inflammation and axonal injury in the central nervous system

Background: Hematoxylin-Eosin (H&E), Cresyl Violet and Bielschowsky Silver Impregnation (BSI) are among the basic histological methods for the evaluation of inflammation and axonal injury in the Central Nervous System (CNS). The usual strategy so far is staining serial sections of CNS separately for H&E and BSI, whilst the simultaneous application of both methods on a single brain section could give more accurate information for both inflammatory and degenerative components. However, the classical protocols for BSI have unstable reproducibility, high staining background and, evidently, hematoxylin is not easily applied.  Materials-Methods: Brain sections from Experimental Allergic Encephalomyelitis (EAE) and Traumatic Brain Injury (TBI) animal models were used. Different pH and incubation periods in the process of BSI staining were investigated to establish a reproducible protocol with minimal background.  Results: The pH of the BSI working solutions strongly affect the final results of the method and the application of specific pHs almost eliminate background up to a point where H&E or cresyl violet counterstaining can easily be applied.  Conclusion: The proposed combination is cost effective, has minimal background and increased reproducibility. It is proposed as a basic screening tool in the study of CNS disorders, namely those where inflammatory process and axonal pathology coexist.