Hrp conjugated goat anti rabbit igg secondary antibody

IHC was performed to analyze HOXA1 expression levels in xenograft tumors were assessed according to a previous study (21 (link)). Briefly, the tumor tissues were dissected and fixed in 4% paraformaldehyde at 25°C overnight, embedded in paraffin and then dissected into sections (5 µm), deparaffinized in xylene, rehydrated with graded alcohols (100, 90, 70 and 50%). Subsequently, the sections were autoclaved for 15 min at 121°C using sodium citrate buffer (0.01 M, pH 7.0) for antigen retrieval. After being blocked with 10% FBS in PBS at 37°C for 30 min, the sections were stained with an anti-HOXA1 primary antibody (1:400 dilution; product code ab230513; Abcam) at 4°C overnight. Subsequently, the sections were incubated with an HRP-conjugated goat anti-rabbit IgG secondary antibody (1:1,000 dilution; product code ab205718; Abcam) for 30 min at 37°C. Then the sections were incubated with DAB for 2 min at 25°C, counterstained with hematoxylin, dehydrated and stabilized with mounting medium (product code ab64230; Abcam). Images were acquired using an IX73 inverted microscope (magnification, ×40; Olympus Corporation). The expression levels of HOXA1 were semi-quantified using Image-Pro Plus software (Media Cybernetics, Inc.).

Total protein from THCA cells was extracted with the help of RIPA lysis buffer (Thermo Fisher Scientific) having protease inhibitor cocktail (Roche, Basel, Switzerland). The quantity of individual protein samples was evaluated availing a BCA test kit (Thermo Fisher Scientific). Through a 12 % SDS-PAGE, protein samples (20 μg) were separated at 150 V for 1.5 h and then transferred onto PVFD membranes (Millipore). Following blocking these with 5 % non-fat milk, the membranes were treated with primary antibodies at 4 °C overnight. The following antibodies were used in this study at a dilution of 1:1000 and were acquired from abcam, Cambridge, UK - Bcl-2 (ab32124), Bax (ab32503), EVA1A (ab216043), and GAPDH (ab9485). The membranes were then treated with hrp-conjugated goat anti-rabbit IgG secondary antibody (abcam, 1:10,000). Finally, each membrane was visualized using a chemical immunogenicity system (GE Healthcare, Chicago, IL, USA), and ImageJ software (NIH, Bethesda, MD, USA) was employed for quantitation of the intensities of the protein bands.

Total protein of tissues and cultured cells were extracted using RIPA buffer (Beyotime Institute of Biotechnology), and was quantified by a BCA Protein Quantification Kit. Total protein (20 µg) was separated using 10% sodium dodecyl sulfonate-polyacrylamide gel (SDS-PAGE; Beyotime Institute of Biotechnology). Then the proteins (20 µg/lane) were transferred onto polyvinylidene difluoride membranes (PVDF; EMD Millipore). Membranes were blocked with 5% BSA (Sigma-Aldrich; Merck KGaA) in TBS with 0.1% Tween-20 (Thermo Fisher Scientific, Inc.) for 1 h at room temperature. Next, the membranes were incubated with primary antibodies against HOXA1 (1:500 dilution; product code ab230513) and GAPDH (1:3,000 dilution; product code ab181602; both from Abcam) overnight at 4°C and then HRP-conjugated goat anti-rabbit IgG secondary antibody (1:5,000 dilution; product code ab205718; Abcam) was added for 2 h at room temperature. Finally, the protein bands were observed using an enhanced chemiluminescence reagent (EMD Millipore) and analyzed by ImageJ software (v1.8.0; National Institutes of Health).