A5971

In a series of cryosections, we investigated if tissue-bound human IgG could be dissociated to address the question of nonspecific binding. Adjacent sections were either treated with 0.5% polyethylene glycol (PEG) or with 0.5 M acetic acid (pH 2.5) before staining with the specific primary AB (human IgG, AQP4, and EAAT2). To exclude or minimize steric hindrance or blocking of the specific ABs by the high concentrations of tissue-bound human IgG we performed double-immunofluorescence stainings on control spinal cord sections using two different commercial anti-AQP4 ABs (rabbit polyclonal, A5971; Sigma Aldrich, 1:500; rabbit polyclonal, sc-20812, Santa-Cruz, CA, USA, 1:500) both directed at the C-terminal domain of AQP4 which is not recognized by the NMO patient IgG fractions (Crane et al., 2011 (link)) in combination with purified NMO-IgG and patient-derived AQP-specific recombinant AB (rAB^AQP4). These stains showed a reproducible binding pattern to predominantly perivascular astrocytic structures. There was no difference in the staining pattern with rAB^AQP4, purified NMO IgG, and both commercial anti-AQP4 AB. Double immunofluorescence staining of naive spinal cord tissue with rAB^AQP4 or purified NMO IgG together with the commercial anti-AQP4 AB gave clear co-labeling (not shown) confirming different binding epitopes of patient and commercial AB and excludes steric hindrance.