Tomato lectin

Transverse sections of the gastrocnemius muscle were immersed in blocking solution (5% normal goat serum in PBS with 0.5% Triton X-100) for 30 minutes at 1, 3, 5, 7 and 9 days after injury, and then incubated overnight at 4 °C with the primary antibody (Tie2, mouse monoclonal antibody, BD Biosciences, Bedford, MA, USA, 1:100; Ki67, rabbit polyclonal antibody, Novus Biologicals, LCC, USA, 1:200; Pax7, mouse monoclonal antibody, R&D Systems, Minneapolis, MN, USA, 1:100; tomato lectin, mouse monoclonal antibody, Vector, Burlingame, CA, 1:200; laminin, rabbit polyclonal antibody, Sigma Aldrich, St. Louis, Mo, 1: 200; eMHC, mouse monoclonal antibody, Developmental Studies Hybridoma Bank, Iowa city, Iowa, USA, 1:200) diluted with PBS. The sections were washed 3 times for 5 minutes each with PBS and then incubated with secondary antibodies (goat anti-mouse IgG-Alexa Fluor 594, goat anti-rabbit IgG-Alexa Fluor 488; Life Technologies Japan, Tokyo, Japan) diluted 1:400 in PBS for 1 h. The sections were washed 3 times for 5 minutes with PBS. Finally, the sections were incubated for 1 min with DAPI (Life Technologies), washed with PBS, and mounted in mounting solution (PermaFluor; Thermo Fisher Scientific Japan, Yokohama, Japan). Positively stained cells were counted in 10 high-power fields (HPFs) using the histological procedures described above (n = 5).

Biotinylated CPI-GC or LPG was prepared using Pierce EZ-Link Sulfo-NHS-LC biotinylation kit as directed by the manufacturer. Bio-layer interferometry was performed on a ForteBio Octet Red (Pall ForteBio Corp., Menlo Park, CA). Streptavidin-coated biosensors were pre-wet in PBS and loaded for 30–60 min in a solution of 50 μg/ml biotinylated CPI-GC. Following a brief wash at pH 3.0 to remove loosely associated lipid, baseline was established by incubation for 5 min in PBS followed by incubation with varying concentrations of lectins or galectins in PBS for 1 h. Dissociation was then examined by incubation for an additional hour in PBS. All steps were performed at 25 °C and at 1000 rpm (except for the loading step, which was done at 0 rpm). For galectin-3, binding was performed in PBS containing 0.005% Tween 20. Steady-state analysis was performed using the ForteBio Data Analysis software (version 6.3). Ricin and tomato lectin were purchased from Vector Laboratories (Burlingame, CA).

Tumor-bearing mice were intraveneously (i.v) injected tetramethylrhodamine 70 kDa dextran (80 mg/kg, Molecular Probes, Waltham, MA, USA) or DyLight 594 labeled lycopersicon esculentum (Tomato) lectin (4 mg/kg, Vector Laboratories, Burlingame, CA, USA), and sacrificed after 40 and 2 min, respectively (20 (link)). Leakage index was defined as the ratio of the signals (pixels) of 70 kDa-dextran to the number of CD31+ vessels.

Animals were deeply anesthetized on P15 with a lethal dose of xylazene/ketamine and perfused transcardially with normal saline, then 4% paraformaldehyde. Brains were sectioned coronally at 6-μm-thick using a microtome. Sections were incubated for 2 h at room temperature in TBS+ 1% Triton-X + 10% donkey serum. Samples were incubated for 24 h at 4 °C with primary antibodies, followed by 2-h incubation at RT with secondary antibodies. All images were captured on a Zeiss confocal microscope (Carl Zeiss, Thornwood, NY, USA). The following primary antibodies were used to detect the following markers: tomato lectin (Vector laboratories, Burlingame, CA, USA), CD 68 (AbS Serotec {1:100} [11 (link), 12 (link)], Raleigh, NC, USA), cleaved caspase 3 (Cell Signaling Technology {1:50}, Danvers, MA, USA), GFAP (Abcam {1:500} Cambridge, MA, USA), Iba1 (Wako {1:400}, Richmond, VA, USA), CNPase (Abcam {1:200} Cambridge, MA, USA), NeuN (EDM Millipore {1:250} Billerica, MA, USA), Olig2 (Santa Cruz Biotechnology{1:50} Dallas, TX, USA), and secondary antibodies (Species specific Cy3 and FITC 1:125 Jackson Immunoresearch, Westgroove, PA, USA). N = 5 animals/group.

Brain sections containing the SNpc were used to perform the immunohistochemical assays. These free-floating sections were first blocked at room temperature for 1 h (PBS 1X, 0.1% of Triton X-100 and 3% of goat serum) and then were incubated with the primary antibodies (α-TH, 1:200, Millipore, Merck Life Science, Madrid, Spain, AB152; α-TH, 1:300, Sigma-Aldrich, Merck Life Science, Madrid, Spain, T2928; α-GFAP, 1:250, Dako, Agilent Technologies, Madrid, Spain, Z0334; α-GFAP, 1:250, Sigma, G3893; α-COX2, 1:200, Cayman Chemical, Ann Arbor, MI, USA, 160106, 160,106; α-TNFα, 1:200, Abcam plc, Cambridge, UK, ab1793) diluted in blocking solution at 4 °C for 24 h. After this, they were incubated for 1 h with the corresponding secondary antibody and 4′, 6-diamidino-2- phenylindole (DAPI; Calbiochem, Merck Life Science, Madrid, Spain 268298) for nuclear staining and diluted in blocking solution at room temperature. Some sections were stained with the fluorescent marker Tomato Lectin (1:150, Vector Labs, Newark, CA, USA, TL1176). Finally, sections were mounted with wet mounting medium (Vectashield; Vector Labs) and observed under an epifluorescence microscope (Nikon 90i).