Brain tissue reactivity patterns of human GABA_AR and GABA_AR-negative mAbs. (A–D) Immunofluorescence stainings of selected human GABA_AR mAbs (green; DAPI nuclei in blue) on fixed murine brain tissue in costainings with commercial antibodies (red/pink) as indicated in the column caption above. (A₁) #113-115 intensively stained hippocampal neuropil throughout CA1 and CA2, most pronounced in stratum oriens (so) with hippocampal fimbria (fi) and stratum radiatum (sr), revealing complete overlap with commercial α1-specific antibody-binding pattern (red; merge in yellow). (A₂) Both antibodies showed distinct staining of the ventral pallidum. (A₃) Higher magnifications in confocal acquisition revealed a somatic staining pattern with complete overlap in the granule cell layer (gcl) of the cerebellum. (B₁) α1γ2-dependent #113-175 stained the olfactory bulb, overlapping with commercial α1-specific antibody, most pronounced in the external plexiform layer (epl) and the molecular cell layer (mcl), whereas weaker in the internal plexiform layer (ipl) and the gcl. (B₂ and B₃) In the cerebellum, the mcl and gcl patterns uncovered different GABA_AR-expressing cell populations, some predominantly labeled by #113-175 (green, black arrowhead), some by commercial antibody (red, white asterisk), and others equally double positive (yellow, white arrowhead). (C₁) #113-201 and commercial α1-specific antibody targeted the epl of the olfactory bulb. (C₂ and C₃) However, #113-201 additionally showed intense binding around blood vessels (bv) and choroid plexus (cp), still detectable at dilutions below the GABA_AR pattern detection (not shown). (D₁) In cerebellar stainings, human mAb GABA_AR–binding pattern (shown for #113-115) clearly distinguished from MAP2-positive dendrites, most pronounced in the mcl (D₂) and from vGAT-highlighted somata of the pcl. (D₃) In magnified confocal images, human mAb visualized GABA_AR clusters throughout the mcl and on a subpopulation of cells within the gcl, shown with a reticular MAP2 costaining. (E–H) Immunofluorescence stainings on unfixed murine brain tissue of human GABA_AR mAb #113-201 or commercial antibodies specific for indicated GABA_AR β-subunits (green; DAPI nuclei in blue). mAb #113-201 bound to the gcl of the cerebellum with expected GABA_AR expression, but additionally also around blood vessels (bv) between the mcl of two neighboring gyri (E₁) and to the choroid plexus (cp) in the ventricles (E₂). Binding of β-subunit–specific antibodies was not detected (F₁, G₁, and H₁) around blood vessels or in choroid plexus (F₂, G₂, and H₂), therefore indicating GABA_AR-independent binding of #113-201 in these locations, as β-subunits are essential for functional GABA_ARs. (I–P) Immunofluorescence stainings of human GABA_AR-negative mAbs (green; nuclei in blue) from GABA_AR encephalitis patient’s CSF repertoire. (I) Germline mAb #113-111 highlighted large bv and cp tissue. (J) #113-126 stained fine vessels, specifically in frontal cortex. (K) #113-128 targeted cp. (L) #113-171, the only GABA_AR-negative mAb derived from an ASC, showed a similar staining pattern as GABA_AR mAbs, including intensive binding to the gcl and mcl molecular but not pcl of the cerebellum. (M) #113-204 was reactive to the mcl and pcl of the cerebellum. (N) #113-210 stained intensively hippocampal neuropil, pronounced in the CA2 and CA3 region, fimbria of the hippocampus (fi). (O and P) #113-220 targeted selectively granule cells in the cerebellum and revealed a punctuated pattern in the lateral septal nuclei. Scale bars indicate 100 µm, 20 µm in the third row (A₃–D₃). alv, alveus; gp, guinea pig; pcl, Purkinje cell layer; rb, rabbit; sp, stratum pyramidale; vp, ventral pallidum.