Hrp conjugated goat anti rabbit igg antibody

Proteins were extracted from the skin healed tissue in the in SDS sample lysis buffer. The samples were heated to 95 °C for 10 min, and 40-μg protein samples were separated on SDS-polyacrylamide gels (5% stacking gel and 12% separation gel). Resolved proteins were then transferred onto nitrocellulose membranes, blocked in 5% nonfat powdered milk for 1 h, and probed with respective antibody against TGF-β1, TGF-β3, Smad2, Smad3, Smad4, Smad7, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as loading control (1:1000 dilution, Abcam, Cambridge, UK) overnight at 4 °C. The blots were blocked in secondary HRP-conjugated goat anti-rabbit IgG antibodies (1:1000 dilution, Abcam, Cambridge, UK) and visualized by chemiluminescent detection substrates (Immobilon western chemiluminescent HRP substrate, Millipore). The densitometric quantification were performed on the protein bands by using AlphaEaseFC software (Alpha Innotech).

As previously stated, RIPA buffer containing proteinase inhibitors was utilized to extract total protein from N2a cells or rat brain cortex [23 (link)]. A BCA assay was applied to measure the lysed protein concentrations. Then, proteins from individual samples prepared above were separated by 10% SDS–PAGE and transferred to PVDF membranes overnight at 4 °C. Then, the membrane was probed with appropriate primary antibodies, namely, DOCK4 (CST; 1:500), Bcl2, Bax, caspase3, and GAPDH (Abcam; 1:1000). Blots were subsequently probed using secondary HRP-conjugated goat anti-rabbit IgG antibodies (Abcam; 1:2000) for approximately 90 min at ambient temperature. An improved chemiluminescence kit (Pierce, Thermo Fisher Scientific, Inc.) was employed to detect proteins.

Protein extracted from cells was lysed in radioimmunoprecipitation (RIPA) buffer, and cell debris was removed by centrifugation. The extracts were quantified using a protein assay and then boiled in SDS gel-loading buffer containing 10% β-mercaptoethanol. Proteins were separated in 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE) gels and then transferred onto immobilon PVDF membranes (Bio-Rad, Hercules, CA), which were probed with rabbit mAbs against mouse β-actin (CST, Danvers, MA), and c-myc and HIF-1α (Wanleibio, Co., Shenyang, China), and then incubated with HRP-conjugated goat anti-rabbit IgG antibody (Abcam, Cambridge, UK) followed by chemiluminescence detection (Champion Chemical, Whittier, CA).

For fibrosis examination, liver tissues were fixed with 10% neutral buffered formalin and embedded in paraffin. Subsequently, 5 μm tissue sections were dewaxed and rehydrated in decreasing concentrations of ethanol and stained with Sirius red (Sigma). The extent of fibrosis was captured under a microscope and quantified by Image software (Carl Zeiss, Germany). For immunohistochemistry analysis, 5 μm tissue sections were boiled in sodium citrate buffer (10 mM, pH 6.0) for 30 min for antigen retrieval and treated with 3% hydrogen peroxide to inhibit endogenous peroxidase activity. The sections were treated with 5% goat serum to block nonspecific binding sites and incubated with primary anti-α-SMA antibody (Abcam) at 4 °C overnight, followed by secondary HRP-conjugated goat anti-rabbit IgG antibody (1:500, Abcam) at 37 °C for 1 h. The color was developed using a DAB mixture (Beyotime). The extent of fibrosis was captured under a microscope (Carl Zeiss Axiostar plus, Germany) and semi-quantified by detecting the average optical density of the histochemical or immunohistochemical reactant area by ImageJ software as previously described [37 (link)].

Dissected L4-5 spinal cords of sham, CCI, LV-NC, and LV-shANXA3 treated rats were cleaned using cold phosphate-buffered saline with 2 mM of EDTA and lysed with a denaturing SDS-PAGE sample buffer via conventional techniques. Protein lysates were divided with a 10% SDS-PAGE and the proteins were shifted onto a PVDF membrane (Millipore, Bedford, MA, USA). The PVDF membranes with proteins were blocked using TBS with 0.1% Triton X-100 and 5% skim milk (overnight; 4°C), and were incubated (overnight; 4°C) with diluted rabbit anti-ANXA3 antibody (1:300, Cell Signal Technology, USA). After TBS cleaning three times, the membranes were incubated (room temperature; 2 h) with diluted (1:5000) HRP-conjugated goat anti-rabbit IgG antibody (Abcam, USA). The protein was inspected using an ECL system (Amersham Biosciences, USA). β-tubulin antibody (Multisciences, China, and 1:2,000 dilution) was probed as a loading control. The Western blots were digitalized using a camera. The ratio of ANXA3 to tubulin was determined by densitometry evaluation. All Western blot analysis (the same biological sample) was repeated at least three times.

Cell lysates were extracted from cultured cells with RIPA buffer (Cell Signaling Technology) supplemented with the protease inhibitors. The lysates were subjected to ultrasonication and centrifugation. The protein concentrations were determined with the BCA Protein Assay Kit (Pierce). Equal amounts (30–40 μg) of proteins samples were separated by 8–12 % SDS–polyacrylamide gel electrophoresis and then transferred onto a nitrocellulose filter membranes (Millipore). After blocking in phosphate-buffered saline/Tween-20 containing 5 % non-fat milk, the membrane was immunoprecipitated with indicated primary antibodies overnight at 4 °C: rabbit polyclonal anti-human HIF-1α antibody (1:1000, Abcam), rabbit polyclonal anti-human Bcl-2 antibody (1:500, Abcam), rabbit polyclonal anti-human Bax antibody (1:500, Abcam), and rabbit polyclonal anti-human β-actin antibody (1:1000, Abcam). The membranes were washed with TBST (3 × 5 min), and then immunoprecipitated with the secondary antibody [HRP-conjugated goat anti-rabbit IgG antibody (Abcam)] for 1 h at room temperature. Subsequent visualization was detected with SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific). Intensities of the bands were normalized to the corresponding β-actin bands.