Nunc maxisorp flat bottom 96 well plate

Owing to the limitation of available serum samples, ELISA was performed with 1% Triton X-100 (Thermo Fisher Scientific, #28314) treated plasma fractions. ELISA was performed in similar conditions as described above with the following modifications. Nunc Maxisorp flat-bottom 96-well plates (Thermo Fisher Scientific, #442404) were coated overnight with 50 μL per well of 0.5 μg/mL of NeutrAvidin protein (Thermo Fisher Scientific, #31050). Blocking was performed for 1 hour with 0.01% polyvinyl alcohol (PVA; Sigma-Aldrich, #341584) in 0.1% PBST (blocking buffer) prepared from stock of 0.5% PVA w/v in distilled H₂O. Peptide coating was performed at 1:2000 dilution for 1 hour. Secondary antibody was incubated for 1 hour in blocking buffer at 1:1000 dilution. Development was performed with 50 μL of TMB and stopped with 50 μL of 0.16 M sulfuric acid.

Nunc MaxiSorp™ flat-bottom 96-well-plates (Thermo Scientific) were coated with F(ab')2 fragment anti-rat or anti-mouse IgG (H+L) (Jackson ImmunoResearch) overnight. Plates were washed with PBS + 0.05% Tween20 and incubated with blocking buffer (PBS containing 0.05% Tween20 and 1% BSA) to block non-specific binding sites. Plasma samples or tissue lysates diluted in blocking buffer were incubated for 2 h or overnight. A standard was generated using respective anti-IL-34 or anti-CSF1R antibodies that were used for in vivo treatment. After washing, plates were incubated with peroxidase-conjugated anti-rat or anti-mouse Fcγ subclass 2a-specific IgG (Jackson ImmunoResearch) for 2 h, then washed and incubated with 1-Step™ Ultra TMB-ELISA Substrate Solution (Thermo Scientific). The reaction was stopped with 2N H₂SO₄ and the signal was measured on a plate reader at 450 nm.
CSF-1 and IL-34 in plasma or brain were measured by commercially available immunoassays (R&D systems), according to manufacturer's instructions.

Nunc MaxiSorp flat-bottom 96-well plates (Thermo Fisher Scientific) were coated by addition of 100 μL of diluted glycoprotein in PBS to each well, and incubation overnight at 4 °C. The plates were then washed with PBST (PBS containing 0.05% Tween 20, pH 7.4) and blocked by addition of 200 μL PBS containing 1% bovine serum albumin (BSA) to each well, and incubation for 1 h at room temperature. Plates were washed 4 times with PBST, after which 100 μL of biotinylated lectin (5 μg/mL in PBS) was added to each well and incubated for 1 h at room temperature. The plates were then washed 4 times with PBST, followed by the addition of 100 μL of horseradish peroxidase-conjugated anti-biotin antibodies (Cell Signaling Technologies, Beverly, MA) to each well and incubation for 1 h at room temperature. The plates were again washed 4 times with PBST, and the 3, 3′, 5, 5′-tetramethylbenzidine (TMB) liquid substrate system (Sigma) was used for colorimetric detection of lectin-glycoprotein complexes. The detection reaction was stopped by addition of 100 μL of 1 M HCl, and the absorbance was measured at 450 nm. Black 96-well plates with clear bottom (Costar) were used for ELISA experiments with fluorescent dye-labeled lectin. The working concentration of DyLight 488 labeled SL2-1 in PBS was 0.9 μg/mL.