Alexa 488 conjugated donkey anti mouse antibody

For double immunofluorescence labeling, six adult GAD67-GFP mice were perfused transcardially with 0.1 M phosphate buffer (PB; pH 7.4) containing 4% paraformaldehyde. Lumbar segments 3–5 of the spinal cord were obtained and postfixed with the same fixative for 4 h, placed in 30% (w/v) sucrose solution in 0.05 M PB solution (PBS; pH 7.4) overnight at 48°C and cut into 25-μm thick sections on a freezing microtome. The sections were incubated overnight at 4°C with a mixture of mouse anti-GFP antibody (1:1,000; Abcam) and rabbit anti-NT antibody (1:500; Abcam). The incubation medium was prepared by 0.01 M PBS (pH 7.4) containing 0.3% (v/v) Triton X-100, 0.12% (w/v) carrageenan, 1% (v/v) normal donkey serum, and 0.02% (w/v) sodium azide (PBS-XCD). After a rinse with PBS, the sections were incubated for 3 h at room temperature in PBS-XCD with a mixture of Alexa488-conjugated donkey anti-mouse antibody (1:500; Invitrogen) and Alexa594-conjugated donkey anti-rabbit antibody (1:500; Invitrogen). The sections were mounted onto gelatin-coated glass slides and cover-slipped with 50% (v/v) glycerol and 2.5% (w/v) triethylenediamine (antifading reagent) in 0.01 M PBS. The sections were observed under a confocal laser scanning microscope (FV-1000, Olympus, Japan) with a confocal depth of 1.0 mm.

The procedure for immunocytochemistry was performed following the previously reported protocols^{28 (link)}. Keratocytes were seeded at a density of 2 × 10⁴ cells per milliliter and grown on 4-well Lab-Tek chamber slides (Nalgene Nunc International, Penfield, NY, USA), and 0, 5, and 10 ng/mL of TGF-β1 were treated for 72 h. Cells were fixed with 3.7% paraformaldehyde for 10 min at room temperature, and permeabilization was conducted using 0.1% triton x-100 for 5 min at RT. Following the washing steps with DPBS, cells were blocked using 1% bovine serum albumin (BSA) in DPBS for 30 min at room temperature. The chamber slides were incubated overnight at 4 °C with rabbit polyclonal anti-α smooth muscle actin (1:1000; catalog number: sc-53142; Santa Cruz, Biotechnology). The chamber slides were then washed with DPBS and incubated with Alexa488-conjugated donkey anti-mouse antibody (1:1000; catalog number: A21202; Molecular Probes) for 2 h at room temperature. Staining for F-actin was executed using tetramethylrhodamine isothiocyanate (TRITC)-conjugated phalloidin (1 µg/mL; Sigma-Aldrich, St. Louis, MO, USA). The counterstaining of cell nuclei was carried out using 4′,6-diamidino-2′-phenylindole (DAPI, 10236276001; Roche Diagnostics GmbH, Mannheim, Germany) with mounting solution. Slides were viewed using a fluorescence microscope.

For NK1-R immunostaining, renal cryostat sections (10 μm thick) on glass slides were fixed in acetone/methanol (1:1) and blocked with PBS containing 3% BSA and 10% rabbit normal serum. Subsequently, slides were incubated with a primary antibody raised against the amino terminus region of human NK-1R (Santa Cruz Biotechnologies, Heidelberg, Germany) and specific markers for macrophages (mouse anti-rat ED1; Serotec, Düsseldorf, Germany) as well as DCs (mouse-anti-rat CD11c antibody; Serotec, Düsseldorf, Germany). Eventually, binding sites of NK-1R were detected using Cy3-conjugated rabbit-anti-goat antibody (1:500; Jackson ImmunoResearch/Dianova). Detection of ED1 and CD11c was performed using an Alexa488-conjugated donkey-anti-mouse antibody (Molecular Probes, Invitrogen, Karlsruhe, Germany).

To estimate the target distribution of BCs using light microscopy, sections were incubated in mouse anti-Kv2.1 (1:1000, 75–014, Neuromab), which was visualized with Alexa488-conjugated donkey anti-mouse antibody (1:500, Molecular Probes). Sections were mounted on glass slides in Vectashield (Vector Laboratories). Images were taken using an A1R or a C2 confocal laser scanning microscope (Nikon CFI Plan Apo VC60X Oil objective, z step size: 0.13 μm, xy: 0.08 μm/pixel). To investigate the number of contacts from a presynaptic BC onto the perisomatic region, terminals on 10–20 randomly chosen neighboring Kv2.1-immunolabeled cells were analyzed. To determine the ratio of perisomatic (i.e. boutons forming close appositions with Kv2.1-labeled profiles) and dendritic (i.e. boutons apposing no Kv2.1-labeled profiles) contacts, targets of 150–200 randomly chosen biocytin-filled terminals were analyzed for each BC.