Anti vgat

For immunocytochemical stainings (ICC) and for Western blots (WB) following primary antibodies were used from rabbit: anti-CDK5 (WB 1:1000, C-8 Santa Cruz), anti-homer1 (ICC 1:1000, Synaptic Systems), anti-VGLUT1 (ICC 1:1000, Synaptic Systems), anti-VGAT (ICC 1:1000, Synaptic Systems), anti-VGAT lumenal domain Oyster550-labeled (ICC 1:200, Synaptic Systems), from mouse: anti-synaptotagmin1 lumenal domain Oyster550-labeled (ICC 1:250, Synaptic Systems), anti-β-tubulin isotype III (WB 1:2000, Sigma), anti-Aβ_17–24 (4G8) (5 μg/ml, Signet), and from guinea pig: anti-synaptophysin (ICC 1:1000, Synaptic Systems). For ICC Alexa Fluor 488- (1:2000), Cy3- (1:2000) and Cy5- (1:1000) fluorescently labeled secondary antibodies were purchased from Jackson ImmunoResearch. For WB secondary antibodies labeled with Alexa Fluor 680 (1:20,000, ThermoFisher Scientific/Molecular Probes) and IRDye 800CW (1:20,000, Rockland) were used.

We used the following MMP inhibitors: FN-439 (180 μM, Calbiochem), UK-356618 (2 μM, Sigma-Aldrich), and SB3-CT (10 μM, Sigma-Aldrich). FN-439 is a broad-spectrum MMP inhibitor that inhibits MMP1, MMP2, MMP3, MMP8, and MMP9 at the tested concentration. UK-356618 inhibits the activity of MMP3 and MMP13 at 2 µM and partially blocks MMP9 (K_i for MMP9 = 840 nM). SB-3CT is a specific inhibitor of MMP2 and MMP9 (K_i for MMP2 = 14 nM; K_i for MMP9 = 600 nM) [20 (link)]. Recombinant human active rMMP3 (Sigma, catalog no. SRP7783) was applied at a concentration of 400 ng/ml.
Anti-gephyrin (catalog no. 147 111, 1:500) and anti-vGAT (catalog no. 131 006, 1:500) antibodies were purchased from Synaptic System. Alexa 488 and Alexa 633 fluorescent secondary antibodies against mouse and chicken antibodies were obtained from ThermoFisher Scientific.

Free-floating sections from cervical, thoracic, and lumbar spinal cord tissue were used for IHC. Sections were first incubated in a blocking solution composed of 1% BSA, 0.5% Triton X-100 and 2% normal goat serum in PBS to reduce non-specific labeling. Sections were then incubated in primary antibodies overnight at 4°C. After multiple washes, sections were incubated in secondary antibodies for 4 h at 4°C. Primary antibodies used were rabbit anti-Polycystin-L (anti-human PKD2L1, 1:1,000 dilution; Millipore, Billerica, MA, USA), guinea pig anti-vesicular GABA Transporter (anti-VGAT, 1:500; Synaptic Systems, Germany) and rabbit anti-GAD65/67 (1:500; Abcam, MA, USA). Secondary antibodies were Alexa Fluor 488- or Alexa Fluor 568-conjugated anti-rabbit (1:500; Life Technologies) and Alexa Fluor 488-anti-guinea pig (1:500; Life Technologies). The same blocking solution as previously was used to dilute the antibodies. PBS was used for washing sections between each step. Sections were mounted on glass slides, cover slipped with Prolong® Gold Antifade Reagent (Life Technologies) mounting medium and then imaged on a Leica SP2 AOBS AOTF inverted confocal laser scanning microscope. Single optical slice images of individual labeled cells were taken using a 63× oil objective.

The anti-ProSAPiP1, anti-LAPSER1 and anti-Shank3 antibodies have been described elsewhere (Wendholt et al., 2006 (link); Schmeisser et al., 2009 (link), 2012b (link)). Furthermore, a polyclonal anti-SPAR antibody directed against amino acids 1461–1735 of this protein was generated for this study based on a previously published protocol (Schmeisser et al., 2009 (link)). The following antibodies were purchased from commercial suppliers: anti-β-Actin (Sigma-Aldrich) anti-β3-Tubulin (Covance), anti-Bassoon (Enzo Life Sciences), anti-PSD95, anti-VGluT1 and anti-VGAT (all Synaptic Systems). Both the full length ProSAPiP1 cDNA sequence (Wendholt et al., 2006 (link)) and RNAi oligonucleotides purchased from Eurofins targeting exon 3 of ProSAPiP1 (5′-GCCTTCAAGCCTGTTGTAC-3′) were cloned into the FUGW vector system. The GFP-SPAR2, GFP-SPAR3 and SPAR-RNAi constructs have been described elsewhere (Richter et al., 2007 (link); Spilker et al., 2008 (link); Dolnik et al., 2016 (link)).

The following mouse primary antibodies were used: anti-GluA1 (NeuroMab 75-327, 1:1000), anti-FAAH (Abcam 1:1000), anti-PSD95 (NeuroMab 75–028, 1:1,000,000) and anti-α-Tubulin (Abcam, 1:50,000). The following rabbit primary antibodies were used: anti-GluA1 phospho-S845-specific (Millipore 1:1000) and anti-CB1 (Cell Signaling, 1:1000). The following guinea pig primary antibodies were used: anti-vGluT1 (Synaptic Systems 135304, 1:11,000), anti-vGAT (Synaptic Systems 131005, 1:1000). The following secondary antibodies were used: goat anti-mouse 680RD (Licor, 1:10,000), goat anti-rabbit 800CW (Licor, 1:15,000), and donkey anti-guinea pig 800CW (Licor, 1:15,000).

Immunostaining was performed as previously reported^{50 (link),51} . After fixation, permeabilization and blocking, autaptic neurons cultures were incubated with the following primary antibodies: anti-MAP2 (guineapig polyclonal, Synaptic Systems, Goettingen, Germany, Cat. No. 188 004, 1:1000 dilution)^{50 (link)}, anti-VGLUT1 (rabbit polyclonal, Synaptic Systems, Cat. No. 135 302, 1:2000 dilution)^{50 (link)}, anti-VGAT (rabbit polyclonal, Synaptic Systems, Cat. No. 131 002, 1:1000 dilution), and anti-tau (mouse monoclonal, Cell Signaling Technology, Beverly, MA, USA, Cat. No. 4019, 1:1000 dilution). Then, samples were incubated with the appropriate species-specific fluorochrome-labelled goat secondary antibodies [Alexa Fluor 488 (Invitrogen, Carlsbad, CA, USA, Cat. No. A11073) for MAP2, Alexa Fluor 594 (Invitrogen, Cat. No. A11037) for VGLUT1 and VGAT, and CF 488A (Sigma-Aldrich, St. Louis, MO, USA, Cat. No. SAB4600042) for tau, 1:400 dilution]. Samples were mounted in mounting medium containing DAPI (ProLong Gold Antifade Mountant with DAPI, Invitrogen, Cat. No. P36931).
Confocal images of neurons were captured as previously reported⁵¹ . Synaptic puncta were analysed according to previously reported procedures^{50 (link)–52 (link)}. Dendritic and axonal morphologies were analysed using NeuronJ, which was accessed as an ImageJ plugin (v1.4.3, Erik Meijering).