Pararosaniline Fixation for Detection of Co-stimulatory Molecules, Cytokines, and Specific Antibody

Abstract

SUMMARYIntegral 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 ␥), 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.

Prevention of Experimental Autoimmune Encephalomyelitis in Common Marmosets Using an Anti-IL-12p40 Monoclonal Antibody

“…To assess whether the biotinylated Ab bound to IL-12p40 produced by astrocytes, tissue sections were double stained using the C8.6 mAb against IL-12p40 and donkey anti-human polyclonal Ab (Sanbio) directed toward glial fibrillary acidic protein characteristic for astrocytes. A combination of HRP and alkaline phosphatase-labeled conjugates was used, giving a red precipitate for 3-amino-9-ethyl-carbazole and a bright blue precipitate using Fast Blue BB base and napthol AS-MX phosphate for alkaline phosphatase, as described in detail previously (32,52).…”

“…IL-12 is expressed at sites where T cells and APC interact (8 -10). In the common marmoset EAE model, such sites are the secondary lymphoid organs and the developing lesions within the CNS white matter (32,52). While microglia is an important source of IL-12 within the CNS (10, 59), astrocytes have also been found to produce IL-12p40 (60).…”

Bacterial peptidoglycan and immune reactivity in the central nervous system in multiple sclerosis

“…Immunohistochemical procedures used for detection of cellular subsets, antigens, antigen specific plasma cells, costimulatory molecules and cytokines in frozen sections have all been described in detail previously (Claassen and Jeurissen, 1998;Schrijver et al, 2000). The frozen sections were fixed in acetone with 0.5% H 2 O 2 for 10 min.…”

Effect of oestradiol and pathogen‐associated molecular patterns on class II‐mediated antigen presentation and immunomodulatory molecule expression in the mouse female reproductive tract

“…Whereas the expression of MHC II by FRT tissues has been documented, the expression of co‐stimulatory molecules is less certain 11,13,14,51,52 . This may be partly because of the low sensitivity of procedures that rely on antibody staining, especially for detecting less stable molecules that are weakly expressed by some APC 53,54 . Using a highly sensitive RT‐PCR analysis, we were able to demonstrate constitutive expression of MHC II, CD40, CD80, CD86 and pIgR in uterine and vaginal tissues.…”