Sc 10741

To investigate the effect of lycopene on the HDM-induced TLR4 activation, cells were pretreated with lycopene for 1 h, before treatment with HDM for 2 h on glass slides, and then fixed with cold 100% methanol. The fixed cells were blocked for 1 h in a blocking solution and then incubated at 20–22 °C for 1 h with a primary antibody against TLR4 (1:100; SC-10741, Santa Cruz Biotechnology, Dallas, TX, USA). After washing with PBS, the cells were allowed to react with a rhodamine-conjugated mouse anti-rabbit IgG antibody (1:200; SC-2492, Santa Cruz Biotechnology) for 1 h. After removal of the excess of secondary antibody, the cells were washed with PBS and covered with Vectashield antifade mounting medium containing 4,6-diamidino-2-phenylindole (DAPI). The preparations were incubated for 30 min to allow for saturation with DAPI. The cells were stained with rhodamine-conjugated antibody and subsequently examined under a laser scanning confocal microscope (Zeiss LSM700, Carl Zeiss Inc., Thornwood, NY, USA) and photographed.

Sections were dewaxed, dehydrated, rehydrated before immunostaining, and sealed with goat serum. Then, antibodies against COX-2 (#12282, CST), GAPDH (sc-25778, Santa Cruz Biotechnology), or TLR4 (sc-10741, Santa Cruz Bio) were added. Slices were dewaxed in xylene and dehydrated in ethanol. After endogenous peroxidase neutralization and the microwave extraction of antigens, slices were blocked in goat serum, then incubated overnight in primary antibodies against COX-2 (#12282, Cell Signaling Technology), GAPDH (sc-25778, Santa Cruz Biotechnology) or TLR4 (sc-10741, Santa Cruz Biotechnology). Sections were washed in PBS and incubated with a universal secondary antibody (PV-6000, Zsbio, Beijing, China) for 30 min, after which 3-3 diaminobenzidine (DAB) and hematoxylin were applied. After washing in PBS containing 0.05% Tween 20, sections were incubated with a general purpose second antibody (PV-6000, Zsbio, Beijing, China) for 30 min. Reaction products were observed using 3-3 diaminobenzidine (DAB) and stained with hematoxylin. Dyed area calculations were performed in Image-pro Plus 6.0 software and the integrated optical density (IOD) analysis of three regression fields was performed. Protein expression was expressed as average density, which was total IOD/area.

Generally, the lysed protein samples were subjected to SDS-PAGE and transferred onto nitrocellulose membranes via electrophoresis, while aliquots of samples were used to determine the protein concentration of each sample using a bicinchoninic acid (BCA) kit (KGPBCA, KeyGEN Biotech, Nanjing, China)^{7 (link),8 (link)}. Subsequently, membranes were incubated in blocking buffer for 2 h and incubated overnight at 4 °C with the following primary antibodies: anti-TLR4 (1:200; SC-10741; Santa Cruz, CA, USA), anti-p-IKKα + β (1:500; ab2064; Abcam, Cambridge, UK), anti-IKKα + β (1:1000; ab178870; Abcam, Cambridge, UK), anti-p-IκBα (1:1000; ab12135; Abcam, Cambridge, UK), anti-IκBα (1:500; ab32518; Abcam, Cambridge, UK), anti-p-NF-κB p65 (1:500; #3033; Cell Signaling Technology, Boston, USA), anti-NF-κB p65 (1:1000; ab16502; Abcam, Cambridge, UK), and anti-HO-1 (1:2000; ab13243; Abcam, Cambridge, UK). GAPDH (1:15,000; ab8245; Abcam, Cambridge, UK) was used as a control on the same membranes. Secondary antibodies were applied, and the bands were detected with West Dura Extended Duration Substrate (Pierce, USA) and X-ray film (Kodak, USA). Then relative density of bands (based on gray value) was analyzed using Bandscan 5.0 software (Glyko, USA) via comparison with GAPDH expression^{7 (link),8 (link)}.

Total proteins were extracted, and quantified using the bicinchoninic acid method. Then, equivalent weights of protein (40 μg/lane) were separated on 10% SDS-PAGE gels and transferred to a nitrocellulose membrane. The membranes were blocked with 5% non-fat milk in Tris-buffered saline and Tween 20 buffer and then incubated with the following primary antibodies: mouse monclonal NF-κB (p65; 1:500 dilution; sc-8008, Santa Cruz Biotechnology) and rabbit polyclonal TLR4 (1:500 dilution; sc-10741, Santa Cruz Biotechnology) at 4°C overnight. Subsequently, after being washed twice with PBS, the membranes were incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit (ZDR-5306) and goat anti-mouse (ZDR-5307) secondary antibodies (1:2,000; ZSGB-BIO, Beijing, China). Specific bands were visualized using an enhanced chemiluminescence detection kit (Immobilon Western Chemiluminescence HRP Substrate; Merck Millipore, Darmstadt, Germany). Optical densities were detected using Quantity One software (Bio-Rad Laboratories, Hercules, CA, USA).