Dylight 649

Dual staining for FISH and lectin was carried out as previously described [30 (link),31 (link)]. Briefly, deparaffinized sections were incubated at 37 °C overnight with Alexa Fluor 488-conjugated gamma-Proteobacteria Phylum GAM42a (5′-GCC TTC CCA CAT CGT TT-3′), which recognizes bacteria belonging to the γ-Proteobacteria class, in hybridization buffer (20 mM Tris-HCl, pH 7.4, 0.9 M NaCl, 0.1% sodium dodecyl sulfate). Sections were rinsed with wash buffer (20 mM Tris-HCl, pH 7.4, 0.9 M NaCl) and PBS twice each, and were co-labeled with fluorochrome-conjugated lectin (Ulex europaeus agglutinin I, UEA1, DyLight^® 649, DL-1068, Vector Laboratories) then mounted with VECTASHIELD. The fluorescence intensity of lectin was quantified using ImageJ software and represented as the integrated fluorescence intensity of the lectin relative to total DAPI values per tissue section. Slides were stained under the same conditions and at the same time.

A double-label immunofluorescence procedure was used to determine whether RET proto-oncogene (RET) expression was affected in dopaminergic neurons by CERE120 therapy. Midbrain sections from each brain were incubated in the first primary antibody (RET, 1:250; polyclonal; R&D Systems) overnight and the donkey anti-goat antibody coupled to DyLight™ 488 (1:200, Jackson ImmunoResearch) for 1 h. After blockade for 1 h, the sections were then incubated in the second primary antibodies (TH, 1:5000) overnight, and the horse anti-mouse antibody coupled to DyLight™ 649 (1:200, Vector) for 1 h. The sections were mounted on gelatin-coated slides, dehydrated through graded alcohol, cleared in xylene, and covered using DPX (Sigma-Aldrich).

Prior to perfusion fixation (approximately 10–15 min), mice were injected intravenously with 100 μl DyLight 649 labeled Lycopersicon esculentum Lectin (Vector Laboratories Cat. No. DL-1178).

Mice (EAE n = 13, control n = 9) were anesthetized with a ketamine-xylazine mix (Ketamine, 100 mg/kg; Xylazine, 20 mg/kg). Shortly before perfusion-fixation, 50 µl of 1 mg/ml Lycopersicon Esculentum lectin conjugated to DyLight649 (DL-1178, VectorLabs) was transcardially injected [5 (link)]. After 5 min of circulation animals were transcardially perfusion-fixed with phosphate buffered saline (PBS) containing 5 IU/ml heparin followed by 4% Paraformaldehyde (PFA, in 0.2 M phosphate buffer, pH 7.4) for fixation of the tissue. Spinal columns were harvested and post-fixed overnight in 4% PFA. The iDISCO+ (immunolabeling-enabled three-dimensional imaging of solvent-cleared organs) protocol was carried out as described by Renier et al. (2016). Briefly, the spinal columns were first decalcified in EDTA (10% w/v, pH 8–9, for 24 h). Next, the tissue was dehydrated in increasing methanol/H₂O series (20%, 40%, 60%, 80%, 100%, 100%, 1 h each), delipidated with methanol/dichloromethane (33%/66% for 3 h) and pure dichloromethane (2 × 15 min), and optically cleared with dibenzyl ether (DBE, 100%) for at least 14 days. Lastly DBE was replaced with ethyl cinnamate (ECi, 100%) at least 7 days prior to imaging.

To screen MAbs specific to OBs, cells (A549, TGF-β1-treated A549, SAOS-2, U2OS, hMSCs, and hFOB) were dissociated by trypsin-EDTA (Welgene) and filtered through a 40-μm strainer and washed by phosphate-buffered saline (PBS, pH 7.4). Cells were resuspended in ice-cold PBA (1% bovine serum albumin and 0.01% NaN₃ in PBS) and incubated with primary antibodies, followed by the incubation with fluorescein isothiocyanate (FITC)-conjugated anti-mouse IgG (Santa Cruz Biotechnology, Santa Cruz, CA), Alexa488-conjugated anti-mouse IgG (Thermo Fisher Scientific, Seoul, Korea), phycoerythrin (PE)-conjugated anti-mouse IgG (Thermo Fisher Scientific), and/or DyLight 650-conjugated anti-mouse IgG (Thermo Fisher Scientific). To analyze only live cells, propidium iodide (PI) (Sigma-Aldrich)-negative cells were gated and analyzed by FACSCalibur (BD biosciences) and Cell Quest Software (BD biosciences). For multicolor flow cytometry, cells were incubated with appropriate primary antibodies for 30 min and incubated with Dylight 649-(Vector Laboratories, Burlingame, CA) or Alexa 488-conjugated anti-mouse IgG (Thermo Fisher Scientific) after osteogenic or adipogenic differentiation of hMSCs. The cells were than incubated with PE-conjugated mouse monoclonal anti-human CD73, CD90, or CD146 antibodies, rabbit polyclonal anti-human CD164, or rabbit polyclonal anti-integrin α2, α3, or αV antibodies.