NMO-IgG/AQP4-Ab were first discovered by means of a standard indirect immunofluorescence (IIF) assay, which used a composite of adult mouse tissues as substrate and which was already well established for the detection of a broad range of other CNS autoantibodies . In this assay, NMO-IgG/AQP4-Ab were identified by their distinctive binding to structures adjacent to the microvasculature, the Virchow-Robin spaces and the pia mater on cerebellum tissue cryosections (Figure 1). Later, several independent studies confirmed IHC-F as a useful tool for the detection of NMO-IgG/AQP4-Ab (Table 1) [1, 51, 102]. One of the major advantages of this type of assay is its broad availability, for it can be performed by all laboratories familiar with IIF, a technique widely used in clinical immunology. Moreover, IHC is the only method that permits the detection of coexisting paraneoplastic or CTD-associated antibodies, which might be of differential diagnostic relevance—in particular in NMO-IgG/AQP4-Ab-negative patients—and must therefore not be overlooked . However, some serious limitations apply. First, results are observer-dependent and thus subjective, ie, they require interpretation by a human rater. This is problematic, as rare sera from non-NMO patients and even healthy controls may show binding patterns that mimic NMO-IgG/AQP4-Ab, eg, anti-endothelial antibodies. In our experience, pre-adsorption of sera with guinea pig liver powder, which results in elimination of most non-CNS-specific antibodies but not of NMO-IgG, can therefore be important to avoid false-positive results , although this procedure might cause some loss of sensitivity. Alternatively, counterstaining of samples with suspected AQP4-Ab positivity with AQP4-specific monoclonal antibodies can be helpful in unclear cases. Secondly, the sensitivity of the IIF assay has been found in independent studies to be much lower than that of some of the recombinant assays described below (Tables 1 and 5) [32, 62, 72, 107, 142, 151, 152]. Third, only semiquantitative results can be obtained (by means of serum titration), and end-titers are again observer-dependent. Finally, testing by IHC-F can be labor-intensive and time-consuming, in particular if including pre-absorption and titration of sera. It may therefore not represent the method of choice if high-throughput analysis is demanded. While binding of circulating NMO-IgG to other tissues in the CNS and in peripheral organs such as kidney, stomach or muscle has been described [68, 90], studies that formally demonstrate a significant increase in sensitivity following from the use of composite tissue substrates are lacking; however, use of composite substrates might potentially increase the specificity of this type of assay, in particular if applied in laboratories not familiar with NMO-IgG testing. From our own experience, mouse cerebellum (as used in most studies) might be preferable to monkey cerebellum; however, two studies that directly compared these two substrates produced conflicting results (Table 1) [26, 92]. Recently, it has been proposed that the fine filamentous white matter staining observed with a majority of NMO-IgG positive sera, in particular on primate tissue, may possibly be useful if applied as an additional positivity criterion [31, 104]. To date, 19 studies have evaluated the originally described IHC-F assay  and 14 studies have reported on independent yet similar IHC-F assays (Table 1). The authors found the assay to be 37.5%–95% (median 61.11%) sensitive for NMO samples and to be 93.33%–100% (median 100%) specific for that diagnosis based on controls with diseases other than NMO or MS and on healthy controls (Table 1). The specificity for NMO vs. MS (not CIS or OSMS) was lower (87%–100%; median 97.67%) (Table 1). However, as discussed above, MS and NMO share common clinico-radiological features and recent studies found that 30%–40% of patients with NMO were initially wrongly diagnosed as having MS in the past [69, 113]; therefore, assessment of assay specificity should not be primarily based on MS controls. Overall, 848 IHC-F results from NMO patients and 2656 from controls other than MS or NMOSD have been reported in the literature (Table 1), 524 (61.8%) and 12 (0.5%) of which were positive, respectively. As a potential confounder, however, it must be kept in mind that some patients may have been tested in more than one study.