647 conjugated secondary antibodies

For hematoxylin and eosin (H&E) staining, liver tissues from sacrificed rats were fixed with 40 g/L neutral formaldehyde, dehydrated with ethanol and xylene, and followed by the standard paraffin-embedding procedure. The 4-μm-thick paraffin sections were cut and subjected to H&E staining.
For immunofluorescence analysis, cryostat sections or cultured cells were fixed in methanol: acetone (1:1) for 30 min at − 30 °C, and then blocked with 10% normal goat serum (Thermo Fisher Scientific) for 1 h at room temperature. The primary antibodies specifically against cytokeratin 19 (CK19; Progen, Heidelberg, Germany), OV-6 (R&D), CD44 (BD Biosciences, Franklin Lakes, NJ), CD133 (Abcam), EpCAM (Abcam), Ki67 (Abcam), desmin (Agilent, Santa Clara, CA, USA), Laminin (Sigma), SE-1 (IBL, Gunma, Japan), CD68 (Abcam), galectin-1 (R&D), or galectin-3 (R&D) were incubated at 4 °C overnight followed by washing three times with PBS. Secondary antibodies included Alexa Fluor 488, 555, or 647-conjugated secondary antibodies (Thermo Fisher Scientific) and were incubated at room temperature for 1 h. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI). The staining was visualized with a Zeiss AxioImager microscope (Carl Zeiss, Jena, Germany).

Embryonic tissues were obtained from mice at E9.5, E10.5 and E11.5 and were processed as in Villacorte, 2016^{62 (link)}. Antigen–antibody complexes were visualized using Alexa 488-, 568- or 647-conjugated secondary antibodies (Invitrogen). Finally, nuclei were stained with Hoechst fluorescent dye (Hoechst 33,258; Sigma) and sections examined with a spinning disk confocal microscope using a Plan Apochromat 100x/1.4 Oil DIC objective (Cell Observer Spinning Disk; Zeiss).
Transgenic mice expressing GFP under the control of Sox9 regulatory elements^{34 (link),35 (link)} were also used for immunofluorescence studies. Tissue samples were extracted at E16.5 or from adult mice, embedded in gelatin and processed as described above. Tissues were also analyzed using a spinning disk confocal microscope (Cell Observer Spinning Disk; Zeiss).
When using cell lines for immunofluorescence, cells were seeded on coverslips and fixed with 4% paraformaldehyde before blocking and incubation with antibodies. Samples were examined with a Leica TCS SP5 confocal microscope (Leica Systems GmbH, Watzlar, Germany).

Harvested tissues were treated as previously described (12 (link)). The following primary antibodies were used: rat anti-mouse CD31 (BD Bioscience), goat anti-mouse Prox-1 (R&D Systems), rabbit anti-mouse LYVE-1 (AngioBio, San Diego, CA, USA), goat anti-mouse LYVE-1 (R&D Systems), or anti-smooth muscle actin coupled to Cy3 (Sigma-Aldrich). The next day whole mounts were incubated for 2 h with AlexaFluor488, 594, or 647-conjugated secondary antibodies (all from Invitrogen). Whole-mount z-stacks were acquired either on a Leica TCS SP8 (Leica Microsystems, Wetzlar, Germany) or on a SP2 AOBS confocal microscope (Leica). Images were acquired using the Leica Application Suite LASX software (version 1.8.0.13370; Leica Microsystems) or the Leica confocal software (version 2.61; Leica Microsystems), respectively. Lymphatic vessels on the pleural side of the diaphragmatic muscle were analyzed as described previously (23 (link)). Per diaphragm two segments of the diaphragmatic muscle were analyzed with ImageJ (NIH, Bethesda, MD, USA) in order to quantify the LYVE-1 positive area, the average branch length and the number of branches, as well as the total vessel length. The number of branch points was quantified manually. All image-based quantifications were performed in a blinded manner.

DAT::Nrxn123WT and DAT::Nrxn123KO mice (P90) were anesthetized using pentobarbital NaCl solution (7 mg/mL) injected intraperitoneally and then perfused intracardially with 20 mL of PBS followed by 30 mL of 4% PFA. The brains were extracted, placed in PFA for 48 hr and then in a 30% sucrose solution for 48 hr. After this period, brains were frozen in –30°C isopentane for 1 min. Forty-µm-thick coronal sections were then cut using a cryostat (Leica CM1800) and placed in antifreeze solution at –20°C until used. For slice immunostaining, after a PBS wash, the tissue was permeabilized, non-specific binding sites were blocked and slices were incubated overnight with a rabbit anti-TH (1:1000, AB152, MilliporeSigma, USA), a mouse anti-TH (1:1000, Clone LNC1, MAB318, MilliporeSigma, USA), a rat anti-DAT (1:2000, MAB369; MilliporeSigma, USA), a rabbit anti-VMAT2 (1:2000, kindly provided by Dr. GW Miller, Columbia University) or a chicken anti-GFP (1:1000, GFP-1020; Aves Labs, USA) antibody. Primary antibodies were subsequently detected with rabbit, rat, or chicken Alexa Fluor-488-conjugated, 546-conjugated, and/or 647-conjugated secondary antibodies (1:500, 2 hr incubation; Invitrogen). Slices were mounted on charged microscope slides (Superfrost/Plus, Fisher Scientific, Canada) and stored at 4°C prior to image acquisition.