Hrp conjugated goat anti rabbit secondary antibody

The fifteen normal canine mammary gland and tumor tissue samples were lysed with RIPA lysis buffer (Beyotime Biotechnology, Shanghai, China), and the protein concentration was determined using a BCA protein assay kit (Beyotime Biotechnology, Shanghai, China). Equal amounts of protein lysate from all samples were separated on a 12–15% SDS-PAGE. Then, the separated proteins were transferred onto PVDF membranes. The blots were blocked with 5% skim milk at room temperature for 1 h and incubated overnight with primary antibodies against IL-6, IL-8 and IL-10 (Dilution, 1:500; Bioss Biotechnology, Beijing, China) at 4 °C. Then, the blots were incubated with HRP-conjugated goat anti-rabbit secondary antibody (ZSGB-BIO, Beijing, China; dilution, 1:2000) at 37 °C for 1 h. The blots were developed using ECL reagent (Tanon Bio, Shanghai, China), and the bands were photographed using a ChemidocXRS system (Bio-Rad, California, USA). β-actin (ZSGB-BIO, Beijing, China; dilution, 1:2000) was used as the loading control. Relative protein levels were quantified using ImageJ 1.48 software (National Institutes of Health, Bethesda, MD, USA).

Immunohistochemistry of paraffin-embedded sections was performed to determine the immunoreactivity of TLR4, NF-κB p65, and TNF-α. The sections were deparaffinized, rehydrated, quenched with 3% H₂O₂ for blocking endogenous peroxidase activity, and washed in PBS before antigen retrieval by microwaving in citrate buffer. Following the application of blocking serum, the sections were incubated in the following primary antibodies overnight at 4°C: anti-TLR4 antibody (1:50, ab 13556, Abcam), anti-NF-κB p65 antibody (1:300, ab16502, Abcam), anti-TNF-α antibody (1:100, ab6671, Abcam). After being washed for 15 min in PBS, the sections were incubated with HRP-conjugated goat anti-rabbit secondary antibody (1:200, ZB02301, ZSGB-BIO) for 50 min at room temperature. DAB was used, and counterstaining was performed with hematoxylin. The slides were then dehydrated and mounted in mounting medium. Microscopy of the sections was performed by a “blinded” pathologist, and the mean density (IOD/area) of identical regions of interest in the hippocampus across eight microscope fields (400× magnification) was determined by Image-Pro Plus 6.0. Nuclear translocation of NF-κB p65 was assessed, and the IOD/nuclear area was compared among the groups.

Total protein lysates were prepared from EC cells and quantified by the Bradford method (5000201, BIO-RAD, USA). Equal amounts of cell lysates (20 μg) were separated by 12% SDS-PAGE and transferred onto nitrocellulose membranes (Merck Millipore Ltd) (1.5 h-2 h for run; 2 h for transfer). Then, the blots were blocked with 5% nonfat milk and incubated with primary antibodies against DUSP1 (1:1000, sc-1102, Santa, USA), p-ERK1/2 (1:1000, #9102, Cell Signaling, USA), E-Cadherin (1:1000, #3195, Cell Signaling, USA), β-Actin (1:2000, #4970, Cell Signaling, USA) at 4°C overnight. Then, the blots were incubated with HRP-conjugated goat anti-rabbit secondary antibody (ZB2301, ZSGB-BIO, Beijing) for 2 h at room temperature and then developed with ECL (P1020, Applygen, China). The band intensities were determined with the Bio-Rad imaging system (Hercules, CA, USA).

Cells were lysed with RIPA lysis buffer (Beyotime Biotechnology). Lysates were cleared by centrifugation at 12,000 g for 5 min at 4 °C and the total protein concentration was determined by BCA Protein Assay Kit (Beyotime Biotechnology). The lysates were separated using SDS-PAGE and transferred onto PVDF membranes. Membranes were blocked in 5% non-fat dry milk in TBST buffer for 1 hour at room temperature. The membranes were incubated with primary antibody followed by HRP-conjugated goat anti-rabbit secondary antibody (ZSGBbio). Bands were visualized using Pro-light HRP Chemiluminescent Kit (Taingen). Primary antibodies against MDR1/P-gp, ERK, p-ERK and p-AKT were purchased from Cell Signaling Technology. β-actin and AKT antibodies were purchased from Bioss.

Recombinant human IL-1β (HZ-1164) was purchased from Proteintech Group (Wuhan, China). Cell counting kit-8 (CCK-8) was purchased from Beyotime Biotechnology (Shanghai, China). Matrigel was obtained from Corning Inc. USA. p38 inhibitor (SB203580) and PI3K inhibitor (GDC-0941) were acquired from Selleck Chemicals (Shanghai, China). Antibodies:VEGFR-1(#13687-1-AP), p38 (#14064-1-AP), MMP-9 (#10375), MMP-2 (#10373-2-AP), GAPDH (#10494-1-AP), and HIF-1α (#20960-1-AP) were obtained from Proteintech (Wuhan, China), c-Fos (#4384), p-c-Fos (S32, #5348), c-Jun(#9165), p-c-Jun (S73, #9164), were purchased from Cell Signaling Technology, p-p38 (#AB4822) and IL-1R1 (#Ab106278) from Abcam USA, p-PI3K (#BS4605), PI3K (#AP0230), Akt (#BS1810), p-Akt (T308, #AA331) was obtained from Bioworld Technology (Nanjing, China), VE-cadherin (#E-AB-12772) from Elabscience. HRP-conjugated goat anti-rabbit secondary antibody was purchased from ZSGB-BIO (Beijing, China).

Total proteins were extracted from cultured cells using RIPA lysing buffer (Beyotime, China), according to the manufacturer’s protocol. Proteins were separated by 10%–12% SDS/PAGE gels and transferred to PVDF membrane (Millipore), which was then blocked by 5% (w/v) nonfat dry milk in TBS-Tween (0.2%) for 1 h. Membranes were probed with rabbit anti-rat α-SMA (1:400, Abcam) overnight at 4°C. After several washes in TBS-Tween, membranes were incubated with goat anti-rabbit HRP-conjugated secondary antibody (1:2000, ZSGB-BIO, China). The subsequent visualization was performed using SuperSignal West Pico Chemiluminescent Substrate (Thermo) by the ChemiDoc XRS⁺ with image Lab software (Bio-Rad).