Hrp coupled secondary antibodies

Samples were prepared according to the NuPAGE Technical Guide of Invitrogen. Briefly, after denaturation in NuPAGE LDS sample buffer with DTT 50 mM for 10 min at 70°C the samples and markers were run on a Novex bis-tris gradient gel (4–12%, Thermo Fischer Scientific) using NuPAGE MOPS SDS running buffer and subsequently blotted on a Novex 0.45 μm nitrocellulose membrane (LC2001, Thermo Fischer Scientific). The membranes were washed and blocked in ReadyTector solution A (CANDOR Bioscience GmbH, Wangen im Allgäu), and the primary antibodies rabbit anti-EAAT2 1:2000 (Abcam, Cambridge, United Kingdom) and mouse anti-GAPDH 1:1000 (Sigma-Aldrich) were directly applied for 1 h at room temperature in solution B that also contained horseradish peroxidase (HRP) coupled to the secondary antibodies against rabbit or mouse, respectively. To detect mYFP, membranes were blocked for 30 min in 1× Roti-block solution (Carl Roth GmbH, Karlsruhe) before applying rabbit anti-GFP 1:1000 (ChromoTek GmbH, Martinsried) over night at 4°C in the same solution. Then the HRP-coupled secondary antibodies (Dianova GmbH, Hamburg) were applied at 1:2500 for 3 h at 4°C. Proteins were detected using respective kits from Biozym Scientific GmbH (Hessisch Oldendorf). Proteins were detected by chemiluminescence and submitted to image analysis with ImageJ.

Neurosphere cells cultured with different growth factor combinations for 3 div were harvested by centrifugation and lysed in lysis buffer (50 mM Tris/Cl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 5 mM EGTA, 1% (v/v) Triton-X100, 0.1% (w/v) deoxycholate, 0.1% (w/v) sodium dodecyl sulfate (SDS), 40 mM sodium fluoride, 1 mM orthovanadate, pH 10) supplemented with the protease inhibitors PMSF (1 mM, MP Biomedicals), IAA (18.5 µg/ml, Sigma-Aldrich), SBTI (10 µg/ml, Sigma-Aldrich), Aprotinin (10 µg/ml, Sigma-Aldrich), Leupeptin (0.5 µg/ml, Sigma-Aldrich), and Pepstatin (0.1 µg/ml, Sigma-Aldrich) for 30 min on ice before the debris was removed by centrifugation. The conditioned medium was directly used for protein analysis. The lysates and supernatants were fractionated on a 4–10% gradient SDS polyacrylamide gel together with the Precision Plus Protein Dual Color Standard (Bio-Rad) and semi-dry blotted to methanol-activated polyvinylidene fluoride (PVDF) membranes. The membranes were blocked and incubated with antibodies for Tnc (polyclonal anti-Tnc, rabbit (batch KAF14), 1:3,000) and α-tubulin (DM1α, 1:10,000, Sigma-Aldrich), probed with appropriate HRP-coupled secondary antibodies (1:10,000, Dianova), and developed with the Clarity Western Blot ECL substrate (Bio-Rad).

Analytical grade chemicals were purchased, if not stated otherwise, from Sigma-Aldrich (MO., USA). The following antibodies were used for immunofluorescence on brain sections: CD68 (1:500; rat monoclonal; clone FA-11 (MCA1957, AbD Serotec)), LAMP1 (1:500; rat monoclonal; clone 1D4B, Developmental Studies Hybridoma Bank (University of Iowa, Iowa City, IA, USA)), and Iba1 (1:500, rabbit polyclonal; GTX100042, Genetex). Fluorophore-conjugated secondary antibodies against the corresponding primary antibody species (AlexaFluor 488, AlexaFluor 594, and AlexaFluor 647) were purchased from Invitrogen/Molecular Probes and were diluted 1:500. HRP-coupled secondary antibodies were purchased from Dianova. The following primary antibodies were used for immunoblotting: TMEM106B (1:1000, rabbit monoclonal;E7H7Z, Cell Signaling Technology), and Na⁺/K⁺-ATPase (1:250, mouse monoclonal; clone a5, Developmental Studies Hybridoma Bank (University of Iowa, Iowa City, IA, USA).