Ab83760

Liver tissues were homogenized and lysed in RIPA containing PMSF (Beyotine). The cellular lysates were clarified by centrifugation at 12,000 rpm for 15 min. Protein concentration in the lysate was determined using the Bradford method. Immunoblotting assays were performed using a standard protocol. Protein samples were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Thereafter, the proteins were transferred onto PVDF membranes. After blocking in 5% non-fat milk, the membranes were probed with primary antibodies against TNF-ɑ (ab199013, dilution 1:1000), IL-6 (ab9324, dilution 1:1000), and HGF (ab83760, dilution 1:1000) (Abcam) and GADPH (EMM0215, dilution 1:1000; Elabscience). Protein bands were visualized using an enhanced chemiluminescence method, and analyzed using ImageJ software. Protein expression was normalized against GADPH. The gray ratio of the target bands to that of the internal reference bands was considered the relative expression of the target proteins.

Immunohistochemical staining was performed by two-step procedure. Upon rehydration as above, the slides were subjected to antigen retrieval by pressure-cooking for 15 minutes. Endogenous peroxidase activity was neutralized using peroxide block placement on the slides for 10 minutes at room temperature. The slides were then incubated with anti-HGF polyclonal antibody (ab83760, Abcam, Cambridge, MA; diluted 1: 200) at 4^°C overnight. This was followed by incubation with peroxidase-conjugated polymer (ChemMate EnVision / HRP; Gene Tech, Shanghai, China) for 30 minutes at room temperature. The chromogen reaction was developed in 3,3′-diaminobenzidine (DAB; Gene Tech, Shanghai, China) tetrahydrochloride for 5 minutes. Finally, hematoxylin was used as a light nuclear counterstain.
The percentage of positive-staining cells were graded on a scale of 0-3, with less than 5% positive-staining cells as grade 0, 5-25% as grade 1, 26-50% as grade 2, and more than 50% as grade 3. The intensity of staining also graded on a scale of 0-2, with negative to weak intensity as grade 0, weak-moderate intensity as grade 1, and moderate to strong intensity as grade 2. Finally, the score of percentage and intensity was multiplied. The final score between 0-2 was determined as low expression, and score higher than 2 was determined as high expression.

Liver tissue was removed and immediately fixed in 10% neutral buffered formalin. It was then embedded in paraffin according to the routine process for light microscopy. The paraffin block was then cut into 4 μm thicknesses by rotary microtome (RM2235, Leica Biosystems, Germany). Antigen retrieval was conducted with sodium citrate buffer (10 mM, pH 6.0) at 95 °C. Heated sections were cooled for 30 min at room temperature. Sections were then incubated in 3% H₂O₂ (#1146, DUCSAN PURE CHEMICALS, Korea) and incubated in 5% bovine serum albumin (BSA, A7906, Merck KGaA, Darmstadt, Germany). Primary antibodies against Cyclin D1 (ab134175, Abcam plc., UK) and HGF (ab83760, Abcam plc., UK) were added. An HRP-conjugated secondary antibody (Thermo fisher scientific, Waltham, MA, USA) was then added. Immuno-detection was conducted with 3,3′-diaminobenzidine (SIGMAFAST™, Merck KGaA, Darmstadt, Germany). Counter staining was conducted using hematoxylin. All procedures were carried out in a humidified chamber to prevent the drying out of tissues. Tissues were observed using a microscope (CKX53, Olympus, Tokyo, Japan). Counts of positive reacted cells and total cells were measured by the color deconvolution tool within TMARKER (Ver. 2.146, open-source software) [62 (link),63 (link)].

sEVs extracted above were added to 5×SDS gel loading buffer [30 mM Tris-HCl (pH 6.8), 2% SDS, 0.05% bromphenol blue, 12.5% glycerol, and 2.5% mercaptoethanol] and then boiled for 30 min. Then 20 μg of sEVs were added into each lane on 10% SDS polyacrylamide gels and the mixtures were resolved for Western blotting analysis. The samples were blotted onto PVDF membranes (Millipore, Boston, MA, USA), which were blocked with 5% milk for 1 hour at room temperature and then probed with relevant primary antibodies of CD63 (ab193349, Abcam, Cambridge, MA, USA), GM130 (ab52649, Abcam, Cambridge, MA, USA), and Alix (sc53540, Santa Cruz Biotechnology, Shanghai, China) at 4°C overnight. Subsequently, membranes were reacted with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature and observed using the ECL kit chemiluminescence reagents (Millipore, Billerica, MA, USA). Similarly, tissue proteins were isolated from hearts with RIPA lysis buffer and 20 μg of proteins per sample were analyzed using primary antibodies against hepatocyte growth factor (HGF, ab83760, Abcam, Cambridge, MA, USA), vascular endothelial growth factor (VEGF, ab64154, Abcam, Cambridge, MA, USA), and platelet derived growth factor BB (PDGF-BB, ab16829, Abcam, Cambridge, MA, USA). Other procedures were the same as described above.

Bones were fixed in 10% neutral buffered formalin for 48 h, decalcified in 10% EDTA for two weeks and stained with hematoxylin and eosin (HE). Lungs were fixed with Bouin’s solution and the number of metastatic foci was macroscopically counted.
For IHC, bones were fixed, decalcified, embedded in OCT compound and sectioned at 10μm thickness using a cryostat. After blocking, sections were incubated with the primary antibodies overnight at 4°C, and secondary antibodies for 60 min, mounted with coverslips in VECTASHIELD anti-fade mounting medium (Vector Laboraories Inc, Burlingame, CA) and observed under TCS SP8 confocal laser scanning microscope (Leica Microsystems, Nussloch, Germany)
Tumors and DRGs were sectioned at 10μm thickness, and incubated with primary antibodies to CD-31 (1:50, Abcam, #ab28364), DeadEnd^™ Colorimetric TUNEL System (Promega, #G7360, Madison, WI), Ki67 (1:400, Cell Signaling, #9129), PGP9.5 (1:200, Abcam, #ab8189), CGRP (1:200 Abcam, #ab36001), TRPV1 (1:1,000 Abcam, #ab31895), peripherin (1:1,000 Abcam, #ab4666), or HGF (1:200, Abcam, #ab83760) overnight at 4°C, and a secondly fluorescent-labeled antibody (1:100) for 60 min or a streptavidin-biotin complex, EnVision HRP (Dako, Carpinteria, CA), for 60 min and visualized using a 3,3-diaminobenzidine (DAB) substrate-chromogen solution (Dako Cytomation Liquid DAB Substrate Chromogen System).

Frozen colon tissues, DPSCs, GFP-DPSCs and HGF-DPSCs were homogenized in lysis buffer containing protease inhibitors. After the lysates were centrifuged at 12,000 rpm for 10 min at 4 °C, the supernatants were collected and the total protein concentration was measured by a BCA protein concentration determination kit (Beyotime, P00125) in accordance with the manufacturer’s instructions. Protein extracts were electrophoresed on 5% SDS-PAGE gels and further transferred to polyvinylidene fluoride (PVDF) membranes. After blocking with 5% skim milk for 1 h, the membranes were incubated with primary antibodies against TNF-ɑ (Santa Cruz Biotechnology, sc-52746, 1:500), TGF-β (Cell Signaling Technology, #3711, 1:500), IL-10 (Abcam, ab33471, 1:1000), IFN-γ (R&D Systems, MAB585, 1:1000), HGF (Abcam, ab83760, 1:500) and β-actin (Abcam, ab8226, 1:1000) at 4 °C overnight. Then, the membranes were washed in TBST three times and incubated with horseradish peroxidase (HRP)-conjugated anti-mouse (Abcam, ab6728, 1:5000) and anti-rabbit IgG (Abcam, ab6721, 1:5000) for 1 h at room temperature. The protein bands were visualized by enhanced chemiluminescence (ECL) solution, and the immunoblotting images were captured by an Omega-Lum G imaging system.