Pararosaniline fixation for detection of co-stimulatory molecules, cytokines, and specific antibody

Integral immunohistochemical analysis of immune responses in frozen sections requires that, in addition to constitutively expressed membrane CD markers, less stable determinants can be reliably visualized. Therefore, we compared the commonly used acetone fixation method with pararosaniline fixation for six determinant categories. These categories included selected constitutively expressed markers, inducible co-stimulatory molecules, pro- and anti-inflammatory cytokines (including the novel cytokine IL-18, also known as IGIF and IL-1 gamma), antigen-specific antibody in plasma cells, bacterial peptidoglycan, and lysosomal acid phosphatase activity. Human spleen and mouse spleen activated by agonistic anti-CD40 antibody or TNP-Ficoll immunization were analyzed in parallel with brain tissue from multiple sclerosis (MS) patients and marmoset monkeys with experimental autoimmune encephalomyelitis (EAE), an animal model for MS. Fixation with pararosaniline resulted in better morphology of all tissues and inhibited endogenous alkaline phosphatase activity in brain tissue. Most determinants could be reliably detected. Staining sensitivity and intensity were markedly increased for selected determinant-tissue combinations, e.g., for IL-4 in human spleen and CD40 in human and mouse spleen. These data show that pararosaniline is a useful alternative to acetone, resulting in superior morphology and specific staining for selected determinant-tissue combinations. This provides additional flexibility for in situ analysis of immune reactivity.