EBER in situ hybridization was performed using the PNA probe, which hybridizes with both EBER1 and EBER2, and the detection kit from DakoCytomation (Glostrup, Denmark), as described previously (35 (link), 37 (link)).
Immunohistochemical stainings for EBV latent (EBNA2) and lytic (BZLF1) proteins and double immunofluorescence stainings for LMP1 and the B-cell marker CD79a and for BZLF1 and the plasma cell marker IgA, IgG, and IgM were performed in sections from paraformaldehyde (PFA)-fixed frozen brain tissue blocks of three MS donors analyzed in this study (MS79, MS92, and MS121), as previously described (19 (link), 35 (link)– (link)38 (link)). For double immunofluorescence staining for LMP1 and LMP2A, brain sections were incubated with anti-LMP2A rat MAb (1:50; clone TP4E11; Ascenion, Munich, Germany) and anti-LMP1 mouse MAb (1:100; clone CS.1-4; DakoCytomation) in phosphate-buffered saline (PBS) containing 1% bovine serum albumin overnight at 4°C, and, after washing with a mixture of Alexa Fluor 488-conjugated donkey anti-mouse IgG (Invitrogen, Eugene, OR) and tetramethylrhodamine (TRITC)-conjugated donkey anti-rat Ig (Jackson ImmunoResearch Laboratories, Cambridgeshire, UK), both diluted 1:300 in PBS containing 3% normal donkey serum. After further washings, sections were mounted with antifade mounting medium containing 4′,6-diamidino-2-phenylindole (DAPI) (Invitrogen).
Immunohistochemical stainings for EBV latent (EBNA2) and lytic (BZLF1) proteins and double immunofluorescence stainings for LMP1 and the B-cell marker CD79a and for BZLF1 and the plasma cell marker IgA, IgG, and IgM were performed in sections from paraformaldehyde (PFA)-fixed frozen brain tissue blocks of three MS donors analyzed in this study (MS79, MS92, and MS121), as previously described (19 (link), 35 (link)– (link)38 (link)). For double immunofluorescence staining for LMP1 and LMP2A, brain sections were incubated with anti-LMP2A rat MAb (1:50; clone TP4E11; Ascenion, Munich, Germany) and anti-LMP1 mouse MAb (1:100; clone CS.1-4; DakoCytomation) in phosphate-buffered saline (PBS) containing 1% bovine serum albumin overnight at 4°C, and, after washing with a mixture of Alexa Fluor 488-conjugated donkey anti-mouse IgG (Invitrogen, Eugene, OR) and tetramethylrhodamine (TRITC)-conjugated donkey anti-rat Ig (Jackson ImmunoResearch Laboratories, Cambridgeshire, UK), both diluted 1:300 in PBS containing 3% normal donkey serum. After further washings, sections were mounted with antifade mounting medium containing 4′,6-diamidino-2-phenylindole (DAPI) (Invitrogen).
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