Gad67

Mice brain were prepared as previously described [63 (link)]. Briefly, mice brain homogenized with RIPA buffer (Thermo Fisher Scientific, Rockford, IL, USA), and incubated on ice for 60 min, and centrifuged at 13,000 rpm for 20 min at 4 °C. An equal amount of total protein (30 μg) was subjected to SDS-PAGE (12%), and the membranes were incubated with the following primary antibodies: CB1 (1:500, Abcam, Cambridge, MA, USA), GAD67 (1:1000, Abcam, Cambridge, MA, USA), β-actin (1:1000, Abcam, Cambridge, MA, USA), and GAPDH (1:500, Cell Signaling Technology, Danvers, MA, USA). The membranes were then incubated with horseradish peroxidase-conjugated anti-rabbit and anti-mouse secondary antibodies (1:5000, Sigma-Aldrich, St. Louis, MO, USA). Immunoreactivity was visualized with an ECL Plus detection system (GE Healthcare, Chicago, IL, USA). The relative density of the protein bands was analyzed with ImageJ.

Hippocampal homogenates were determined using Bio-Rad DC protein assay kit (BIO-RAD, Hercules, CA, USA) according to the manufacturer’s protocol. For immunoblotting analysis, 30 μg hippocampus proteins were resolved over 12% Tris-glycine polyacrylamide gel and then transferred onto polyvinylidene fluoride membrane (Merck Millipore, Burlington, VT, USA). The blots were blocked using 5% nonfat dry milk, and these immunoblots were probed using the primary antibodies Tyrosine hydroxylase (EMD Millipore, Darmstadt, Germany), GAD-67 (Abcam, Cambridge, UK), CREB (Cell signaling, Danvers, MA, USA), phospho-CREB (EMD Millipore), GAPDH (Cell signaling) and secondary horseradish peroxidase conjugate (Amersham Life Science Inc., IL, USA). The proteins were detected by chemiluminescence using the ECL kit (Amersham Life Science). The relative density of the protein bands was scanned and quantified by ImageJ (Wayne Rasband, National Institutes of Health, Bethesda, MD, USA).

IHC was performed on serial section slides in two independent multiplexed batches. The first batch followed the sequence mouse anticalcium/calmodulin-dependent protein kinase II alpha (MA1-048; Invitrogen), rabbit antisomatostatin (T-4102; Peninsula Laboratories), rabbit anti-GFP (ab290; Abcam), and mouse anti-PV (PV235; Swant). The second batch followed the sequence, mouse anti-glutamate decarboxylase (GAD67, ab26116; Abcam), mouse anti-fox-3 (NeuN, MAB 377; Millipore), and rabbit anti-GFP. After primary and secondary antibody incubation, slides were stained with DAPI. Whole slide images were captured at 20 × using an Akoya Biosciences Polaris instrument. Image exposures for each fluorochrome were constant for all slides. Detailed image analysis is described in the Supplementary Materials.

The 40-μm floating sections (series of every 12th section from the hippocampus) underwent IFA as described (4) and were incubated with the antibodies β-catenin (BD; 1:500), BCAS2 (Proteintech, Rosemont, IL, USA; 1:10000), Cre (Abcam, Cambridge, UK; 1:500), Sox2 (Abcam; 1:500), BrdU (Abcam; 1:500), NeuN (Millipore, NJ, USA; 1:1000), DCX (Millipore, Darmstadt, Germany; 1:500), GFAP (Sigma; 1:500), GAD67 (Abcam; 1:250), and cyanine Cy3 goat anti-mouse IgG, Alexa Fluor 488 donkey anti-rabbit IgG, Alexa Fluor 647 goat anti-rat igG, Alexa Fluor 488 donkey anti-rat (all Jackson ImmunoResearch PA, USA; 1:1000) (Additional file 2: Table S1). Images were captured by using a Leica TSC SP5 confocal microscope.

Immunofluorescence staining was performed using the method described in our previous publication^{4 (link),24 (link)}. The primary antibodies were: rabbit anti-GHRH (1:100, catalog No: ab187512, Abcam, Cambridge, MA, USA), mouse anti-glial fibrillary acidic protein (GFAP) (1:50, catalog number BM0055; Boster Bioengineering, Wuhan, China), mouse anti-Gephyrin (1:50, Abcam, Cambridge, MA, USA), guinea pig anti-microtubule-associated protein 2 (MAP2) (1:200, Sysy, Goettingen, Germany), mouse anti-glutamate decarboxylase 67 (GAD67) (1:100, Abcam, Cambridge, MA, USA), and guinea pig anti-vesicular GABA transporter (VGAT) (1:200, Sysy, Goettingen, Germany). The secondary antibodies were: an Alexa Fluor 488-conjugated goat anti-rabbit IgG antibody (1:50, Zhongshan Golden Bridge, Inc., Beijing, China), Alexa Fluor 594-conjugated goat anti-mouse IgG antibody (1:200, Zhongshan Golden BridgeInc., Beijing, China), and Alexa Fluor 633-conjugated goat anti-guinea pig IgG antibody (1:50, Abcam, Cambridge, MA, USA). Finally, the samples were treated with 4′,6-diamidino-2-phenylindole dihydrochloride (Sigma, St. Louis, MO, USA) for 5 min to identify the nuclei. Immunofluorescently labeled sections were examined with a laser scanning confocal microscope (Leica Microsystems, Wetzlar, Germany) and an Olympus IX 70 inverted microscope (Olympus America, Melville, NY, USA).

Human GAD65 (Diamyd, Stockholm, Sweden), GAD67 (Abnova, Taipei, Taiwan), and GABARAP (Abnova) recombinant proteins were electrophoretically separated and transferred to a polyvinylidene difluoride membrane and strips incubated with the patient's serum (1:1000 dilution), GAD65 (GAD-6, Hybridoma Bank, Iowa City, IA), GAD67 (Abcam, Cambridge, UK), or GABARAP (Abcam) commercial antibodies followed by biotinylated goat antihuman IgG or horse antimouse IgG (Vector Laboratories, Burlingame, CA) and developed with diaminobenzidine tetrahyrochloride [15 (link)]. In some experiments we used an enhanced chimioluminiscence technique (ECL Western Blotting System) following manufacturer’s instructions. The specificity of GAD65 and GAD67 commercial antibodies was confirmed with immunoblots with recombinant proteins and also with HEK293 cells (see below) expressing the corresponding antigens.

Immunocytochemistry/immunofluorescence was done as described previously [62 (link)]. The cultured cells were incubated with the following primary antibodies: β-tubulin III (TUJ1) (1:300, Abcam, Cambridge, MA, USA), CB1 (1:300, Abcam, Cambridge, MA, USA), GAD67 (1:300, Abcam, Cambridge, MA, USA), c-Myc (1:300, Abcam, Cambridge, MA, USA), and GFP (1:300, Abcam, Cambridge, MA, USA) at 4 °C overnight. Then, cells were incubated with secondary antibodies conjugated to Alexa Fluor 405, 488, or 594 (1:500, Invitrogen, Carlsbad, CA, USA) at RT for 1–2 h. Finally, cells were incubated with DAPI (2 mg/mL stock, 1:1000, Sigma-Aldrich, St. Louis, MO, USA) at RT for 5 min. Images were captured using a Leica DM5500B microscope and Leica DFC495 camera (Leica Microsystems, Wetzlar, Germany). LAS-AF (version 3.1.0 build 8587, Leica Microsystems, Wetzlar, Germany) software was used to merge single monochromatic fluorescent micrographs.