Vimentin antibody

A total of 56 archived gastric cancer tissues were fixed with 4% paraformaldehyde, then paraffin-embedded, sectioned (5mm), deparaffinized, rehydrated, and antigen extracted. The tissue part was used to incubate the CAS blocking buffer and subsequently used to incubate the main antibody (E-Cadherin Antibody, Rabbit Polyclonal, PROTEINTECH, Catalog number: 20874-1-AP. Vimentin Antibody, Rabbit Polyclonal, PROTEINTECH, Catalog number: 10366-1-AP. Vinculin Antibody, Rabbit Polyclonal, Affinity Biosciences Cat# AF5122, RRID:AB_2837608). It was then overnighted at 4° C and DAKO envisio-hrp (DAKO) was used to check the reactivity afterward. By multiplying the percentage of positive cells (P) by the intensity (I), the degree of immunostaining is scored according to the following formula: H = P × I. The range of P is 0 to 4 (5% scored 0; 5-25% scored 1; 25–50% scored 2; 50–75% scored 3; 75–100% scored 4). The range of I is 0 to 3 (0, no staining; 1, weak; 2, medium; 3, strong). The staining index score was calculated by multiplying P by I, and the resulting range is 0-12. The staining index score is defined as followed: 0-3 as negative; 4-12 as positive.

Approximately 2 × 10⁵ primary lung fibroblasts cells were cultivated in a 24-well plate. After transfection and TGF-β1 treatment, these cells were rinsed with cold phosphate-buffered saline (PBS) for three times and fixed in 4% paraformaldehyde for 30 min. The cells were rinsed with PBS for three times, incubated with 0.4% Triton X-100 for 1 h at room temperature, and blocked with 50% normal goat serum for 1 h at 37°C. Afterward, α-SMA antibody (1:200, Abcam, U.S.A.) or Vimentin antibody (1:200, Proteintech, Wuhan,China) was added dropwise into each well at 4°C overnight. Subsequently, incubated with FITC-conjugated goat anti-mouse or goat anti-rabbit antibody for 1 h. All operating steps were conducted in the dark because fluorescent secondary antibodies were added. After rinsing with PBS for three times, the nuclei were stained with DAPI (Roche Molecular Biochemicals, Basel, Swizerland) for 5 min at room temperature. Immunofluorescence was analyzed under a fluorescence microscope (Olympus, IX73, Japan).

rGO was purchased from the JCNANO Tech Co. Ltd (Nanjing, Jiangsu, China). Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DME/F-12) and phosphate-buffered saline (PBS) solution were obtained from HyClone (Logan, Utah, USA). Fetal bovine serum (FBS)was from Gibco Invitrogen (CA, USA). 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium(MTS) was obtained from Promega (MO, USA). 24-well Transwell with 8-μm pore size polycarbonate inserts were purchased from Corning Incorporated (NY, USA). Trizol reagent was from Invitrogen Inc. (Carlsbad, CA, USA). The PrimeScript® RT Master Mix Perfect Real-Time kit and SYBR® Premix Ex TaqTM II kit were from Takara (Dalian, China). RIPA buffer and BCA protein assay kit were purchased from Thermo Scientific (Rockford, IL, USA). Monoclonal β-actin, polyclonal Rab5a, Rab7a, E-cadherin, β-catenin and Vimentin antibody were purchased from Proteintech (Rosemont, IL, USA). Goat anti-rabbit IgG conjugated with 15 nm gold particles were from Abcam (Cambridge, MA, USA).

The total protein of cell or tissue lysate was isolated as previously described [45 (link)]. Cells were harvested in radioimmunoprecipitation (RIPA) assay buffer(Santa Cruz Biotechnology,#sc-24948). Protein content was quantified using the Bradford assay with BSA as standard (Bio-Rad), and 30μg of total protein lysate were separated on SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membrane. After blocking (5% non-fat milk in PBS, 1 h at RT), membranes were incubated at 4°C overnight with the following primary antibodies: Hsp60 antibody (Affinity, #AF0184,1:1000), β-actin monoclonal antibody (Kangchen, #KC-5A08,1:5000), AFP antibody(Abcam, #3980, 1:1000), E-cadherin antibody (proteintech,# 20874-1- -AP,1:1000), Vimentin Antibody (proteintech, #10366-1-AP,1:1000), N-Cadherin antibody (Abcam, #ab18203) and COX4 antibody (Abgent, #ALS12730,1:1000). After three washes with PBS, membranes were incubated with peroxidase-labelled secondary antibodies (Merck Millipore, 1:5000) at 37° C for 1 h. Immunoblots were developed using the enhanced chemiluminescence reagent (Pierce, #32209) .

Western blotting was performed in accordance with the standard protocols. In brief, cellular proteins were extracted using the RIPA lysis buffer, separated on SDS-PAGE gels, and transferred onto NC membranes (Millipore, Billerica, USA). After incubated in primary and second antibodies separately, proteins were detected by ECL reagent (Millipore, USA) and imaged with FluorChem Q (Protein simple, USA). The following antibodies were used for western blotting: PKM2 antibody (Proteintech, China, 1 : 1000, 15822-1-AP), vimentin antibody (Proteintech, China, 1 : 1000, 10366-1-AP), E-cadherin antibody (Proteintech, China, 1 : 1000, 20874-1-AP), N-cadherin antibody (Proteintech, China, 1 : 1000, 22018-1-AP), and GAPDH antibody (ZSGB-bio, China, 1 : 1000, TA-8).