Horseradish peroxidase conjugated goat anti rabbit igg antibody

A peptide corresponding to residues 247–266 (HGPDPRDRPLSDGQIKTIET) of TonB2 was synthesised and used as an antigen to obtain antisera against TonB2 in rabbits (Cosmo Bio, Inc.). Anti-TonB2-peptide antibodies were obtained by purification of the antiserum using peptide affinity column chromatography (Cosmo Bio, Inc.). Total membrane fractions were prepared as described previously from SYK-6 cells incubated in LB for 20 h with or without 100 µM DIP^{13 (link)}. When total membrane fractions were prepared from the tonB2-complemented ∆tonB2, cells were incubated in LB containing Km and 1 mM m-toluate. TonB2 was detected by western blot analysis using anti-TonB2 antibodies (0.09 µg/ml) as described previously^{13 (link)}. Horseradish peroxidase-conjugated goat anti-rabbit IgG antibodies (Invitrogen, 0.2 µg/ml) were used as the secondary antibodies. Protein concentrations were determined by the Bradford method using a Bio-Rad protein assay kit or Lowry’s assay with a DC protein assay kit (Bio-Rad Laboratories). TonB2 was detected using the ECL Western Blotting Detection System (GE Healthcare) with a LumiVision PRO image analyser (Aisin Seiki Co., Ltd).

The extraction and quantification of total protein were carried out from 1 g of frozen cassava roots according to Maass et al. [88 (link)] with modifications of Cuellar et al. [89 (link)]. Proteins were resolved on a 12% SDS-polyacrylamide gel and blotted to 0.45 μM PVDF membranes (ThermoFischer). The blotting was conducted in a Mini Trans-Blot^® Cell (Bio-Rad) apparatus. Membranes were blocked with 5% skim milk powder in Tris-buffered saline, washed, and incubated in Tris-buffered saline plus 0.1% Tween 20 with either the polyclonal antibodies anti-PSY [56 (link)] or anti-OR [51 (link)]. After washing, the membranes were incubated for 1 h with a horseradish peroxidase- conjugated goat anti-Rabbit IgG antibody (Invitrogen, Cat.# G-21234) in a 1% skim milk solution in Tris-buffered saline plus 0.1% Tween 20. The membranes were washed and immunoblots were developed with Pierce^™ ECL Western Blotting Substrate (ThermoFischer). After striping the membrane with peroxidase [90 (link)], an anti-actin antibody (Sigma-Aldrich, Cat.#A0480) and a horseradish peroxidase-conjugated goat anti-Mouse IgG antibody (Invitrogen, Cat.# G-21040) were used to reprobe the immunoblot. Protein signals were quantified with ImageJ [91 (link)].

For cross-sectional observations, the harvested cell sheets, resected tumors and organs were fixed with 4% paraformaldehyde and embedded in paraffin. The specimens were sliced into 5 μm sections, followed by haematoxylin and eosin (HE) staining and Masson staining. For immunohistochemical staining, the sections were blocked with 1% bovine serum albumin (BSA) and 0.5% Triton-X100, then treated with desmin, vimentin, CK-8, CD31 and type I collagen IgG antibody (rabbit anti-human, 1:500, Abcam, Cambridge, MA, USA) at 4 °C overnight. After washing with PBS, the sections were incubated with a horseradish-peroxidase-conjugated goat anti-rabbit IgG antibody (1:1000; Invitrogen, Carlsbad, CA, USA) for 1 h at room temperature. Finally, the sections were stained with 3,3 N-diaminobenzidine tertrahydrochloride (Sigma-Aldrich, USA) and counterstained with hematoxylin. Images were captured using an upright metallurgical microscope (Olympus, Japan).

The total protein of the spinal cord tissues was extracted using RIPA lysis buffer. Total proteins of each group (40 μg) were resolved in 8% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto polyvinylidenefluoride (PVDF) membranes (Millipore, Bedford, MA). After the membranes were blocked in5% non-fat milk at room temperature for 30 min, they were incubated with anti-AQP4 (ab46182, Abcam, Cambridge, MA), anti-NKCC1 (ab59791, Abcam), or anti-β-actin (A2066, Sigma, St Louis, MO) antibody at 4°C overnight. After 3 washes with PBST, the membranes were incubated with proper horseradish peroxidase-conjugated goat anti-rabbit IgG antibody at room temperature for 60 min, and visualized with the enhanced chemiluminescence (ECL) substrate (ThermoFisher, Shanghai, China). The images were scanned and analyzed with ImageJ (NIH, Bethesda, MD).

Platelets were lysed in 1% Nonidet P-40, 150 mM NaCl, and 50 mM Tris/HCl, pH 7.4, containing 1 mM EGTA, 1 mM sodium orthovanadate, and cOmplete Protease Inhibitor Cocktail (Roche). SDS-PAGE buffer was added to lysates in the presence of 1% β-mercaptoethanol. Proteins were resolved by SDS-PAGE following quantification by Bradford protein assay and transferred onto PVDF membrane. After blocking overnight with 1% BSA in 0.2% Tween-20, 100 mM NaCl, and 20 mM Tris/HCl, pH 7.4, membranes were probed with rabbit antibodies directed against total or phosphorylated STAT3 (Tyr705) and STAT5 (Tyr694 in STAT5A; Tyr699 in STAT5B) (Cell Signaling), JAK2, β-actin, or GAPDH, followed by secondary horseradish peroxidase-conjugated goat anti-rabbit IgG antibody (Thermo Fisher Scientific). Detection was performed by enhanced chemiluminescence.

Western analysis on proteins in liver lysate (20 μg/lane) was performed as previously described.²⁵ (link) LDLR and beta-tubulin were detected with rabbit anti-LDLR antibody (ab52818; Abcam, Cambridge, MA, USA) and rabbit anti-beta-tubulin antibody (ab6046; Abcam), respectively. Bound primary antibody was detected with horseradish peroxidase-conjugated goat anti-rabbit IgG antibody (Thermo Fisher) and SuperSignal West Pico Chemiluminescence Substrate (Thermo Fisher).