Hrp conjugated goat anti rabbit antibody

Collected normal and ectopic endometrial tissues were fixed with 4% formalin (Biotech Well) for 12 h at 4 °C, embedded in paraffin and cut into 5-µm-thick slices. Then, the specimens underwent dewaxing, dehydration and antigen retrieval, and they were further incubated with primary antibodies against PTGIS and HIF-1α (Abcam, UK) in a humidified box overnight at 4 °C. The following day, the specimens were washed with PBS 3 times and then incubated with an HRP-conjugated goat anti-rabbit antibody (Abcam) for 30 min at room temperature. Then, all slides were reacted with DAB substrate and hematoxylin dye reagents (Biotech Well). Rabbit-derived isotype-matched immunoglobulin G was incubated with slides used as negative controls. Detailed information on the antibodies and reagents is listed in Supplementary Table 2. Finally, the specimens were observed and imaged randomly under a microscope. The staining intensity of the slides was examined and verified independently by two observers, and scores were calculated as the percentage of positive immunoreactive cells. No staining was recorded as no staining; staining of less than 10% was defined as weak staining; staining of 11–50% was defined as moderate staining; and staining of more than 50% was defined as strong staining.

Total protein was extracted from the cells using the M-PER mammalian protein extraction reagent or from tissues using the T-PER tissue protein extraction reagent (Pierce, IL, USA). Equal amounts of protein (12 μg per lane) as estimated by a bicinchoninic acid protein assay kit (Pierce, IL, USA) were separated by 11% SDS-PAGE and then transferred onto nitrocellulose membranes. The blots were probed with rabbit monoclonal antibodies against UbcH10 (1:200), KIAA0101 (1:500), BubR1 (1:600), Mad2 (1:250), CDC25(1:400), CDK1 (1:300), CDK7 (1:500), CyclinB (1:350) and β-actin (1:1000) (Abcam, CA, USA), followed by incubation with secondary HRP-conjugated goat anti-rabbit antibody (Abcam, CA, USA). After a washing step, the bands were detected by chemiluminescence and imaged on X-ray films. β-actin was used as an endogenous reference for normalization.

Cell lines were purchased from American Type Culture Collection (ATCC number: HTB-26™)(Manassas, VA, USA). Cells were cultured in DMEM media supplemented with 10% FBS, 1% penicillin/streptomycin and 2mM L-Glutamine. Cells were maintained in a humidified incubator at 37°C with 5% CO₂ atmosphere. LC3I/II antibody, HRP-conjugated Goat Anti-Mouse antibody, HRP-conjugated Goat Anti-Rabbit antibody were purchased from Abcam (Cambridge, UK). Anti-GAPDH antibody, anti-caspase-3, were obtained from Cell Signaling Technology (Leiden, The Netherlands). Methylthiazolyldiphenyltetrazolium bromide (MTT) was purchased from Sigma-Aldrich (Schnelldorf, Germany). PgE₂ and NO colorimetric ELISA quantification kits were purchased from Cayman Chemical (Ann Arbor, Michigan, USA). MMP-2 and MMP-9 ELISA quantification kits were obtained from R&D Systems (Minneapolis, MN, USA).

DF-1 cells were harvested at 48 hpi and lysed on ice for 10 min using a cell lysis buffer (Beyotime Biotechnology, Shanghai, China) following the manufacturer’s specifications. After centrifugation at 14,000 × g for 5 min at 4°C, the supernatant was collected and then assayed for total protein content using a BCA Protein Quantification Kit (TransGen Biotech, Beijing, China), and 20 μg of total protein was subjected to 8% SDS-PAGE and transferred to a PVDF membrane (Millipore, United States). PVDF membranes were blocked using 5% skim milk for 1 h at room temperature, incubated with mouse anti-ENV mAb JE9 (Qin et al., 2001 (link)) or rabbit anti-ACTB antibody (Abcam, United Kingdom) at 4°C overnight, washed with Tris-Buffered Saline with Tween 20 (TBST) for three times, then incubated with HRP-conjugated goat anti-rabbit antibody (Abcam, UK) or goat anti-mouse antibody (Abcam, UK) at room temperature for 2 h, and detected with SuperSignal West Pico PLUS chemiluminescence substrate after TBST cleaning for three times. Exposure development was performed using a chemiluminescence imaging system (Azure Biosystems, United States).

The expression of IGHG1, AKT, p-AKT, and vascular endothelial growth factor (VEGF) were analyzed in cell lines and breast cancer tissues [13 (link)]. Cells were lysed and protein levels were assessed via BCA assays (ThermoFischer Scientific cat: 23225). Proteins were resolved using 10% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes (Beyotime, Shanghai, China). Membranes were blocked in Tris buffer containing 5% skimmed milk and probed with the following primary antibodies: anti-IGHG1 (ab 283421, Abcam, China), anti-AKT (ab 8805), anti-p-AKT (ab 81283), anti-β-actin (ab 8227), and anti-VEGF (ab 53465) at 4 ^∘C followed by labeling with HRP-conjugated goat anti-rabbit antibodies (1:1,000; ab 7090) for 2 h at room temperature. Bands were visualized using chemiluminescence detection kits (Thermo Fisher Scientific, Inc., USA).