Anti ho 1 antibody

HSC and HaCaT cells were cultured in Dulbecco's modified Eagles' medium (DMEM, Mediatech), with phenol red, supplemented with 10% v/v fetal bovine serum (FBS, Mediatech) and 1% antimycotic solution (Mediatech) in a 37 °C, 5% CO₂ humidified incubator. For the PERS studies, the mammalian cells were grown on glass coverslips in complete growth medium in an incubator at 37 °C for 24 h. Afterwards, the cells were incubated with 0.075 nM of PEG/RGD/NLS-functionalized nanoparticles (AuNCs), diluted in supplemented DMEM cell culture medium, for 24 h. The concentration of the nanoparticles was determined by using the data obtained from an axial inductively coupled plasma (ICP) atomic emission spectrometer machine. The cells were then synchronized in the G1 phase by serum starvation.44 (link) Subsequently, they were released into the complete medium before PERS experiments. The cell viability assay was conducted by using the XTT cell viability assay kit (Biotium, Inc.) by following the manufacturer's protocol. Anti-HO-1 antibody was purchased from abcam and the GAPDH, secondary mouse and rabbit antibodies were from Santa Cruz Biotechnology.

For retinal cryosections, eyes were enucleated and embedded in optimal cutting temperature compound overnight. Eight-µm serial cryosections were prepared. The slices were probed with primary antibodies (1:100) at 4°C overnight followed by incubation with fluorescence-labeled secondary antibodies (1:1,000 for1 hour). For retinal wholemounts, eyes were fixed in 4% paraformaldehyde for 30 minutes and the retinae were carefully removed. After incubated with primary (1:100, 4°C, overnight) and respective secondary antibodies (1:1000 for 1 hour), and counterstained with 4′6-diamidino-2-phenylindole (DAPI, 1:1000 for 5 minutes), the retinae were washed extensively and flat-mounted on the slides. The primary antibodies include Isolectin (Invitrogen, Waltham, MA, USA), anti-Nrf2 antibody (Abcam, Cambridge, United Kingdom), anti-HO-1 antibody (Abcam), anti-Iba1 antibody (Wako Chemicals, Osaka, Japan), anti-GFAP antibody (Abcam), anti-CD86 antibody (Abcam), and anti-CD206 antibody (Santa Cruz Biotechnology, Dallas, TX, USA). The sections and flat mounts were observed using a confocal microscope (Carl Zeiss LSM700-ZEN 2009, Germany) or an automated upright fluorescence camera (Leica DM4000B, Solms, Germany). Areas of neovascularization were assessed using Image-Pro Plus 6.0 (Media Cybernetic, Rockville, MD, USA).

As outlined in previous publications [30 (link)-32 (link)], HCECs exposed to A. fumigatus for 8 h were lysed in a mixture of RIPA buffer (Solarbio, Beijing, China), phenylmethanesulfonyl fluoride (Solarbio), and phosphatase inhibitor (MCE; mixing ratio: 98:1:1) for 2 h. The protein concentration was quantified by bicinchoninic acid (BCA) assay (Elabscience, Wuhan, China). The protein samples separated by 10% sodium salt -polyacrylamide gel electrophoresis (SDS-PAGE) were transferred onto polyvinylidene difluoride membranes. The membranes blocked with blocking buffer were incubated with anti-β-actin antibody (1:3000; Elabscience) and anti-HO-1 antibody (Abcam;1:10000) at 4 °C overnight. After being washed in PBST and incubated with the corresponding secondary antibodies for 1 h, the bands on the membranes were detected with enhanced chemiluminescence (ECL) reagents (Biorad, CA, America). A quantitative grayscale analysis of the western blot bands was performed using Image J.

Retinal and cellular protein was harvested and homogenized in lysis buffer containing protease and phosphatase inhibitor mini tablets (Thermo Fisher Scientific, Waltham, MA, USA). The protein concentration was determined with bicinchoninic acid protein assay. Equal amounts of protein were loaded and Western blotting was performed as previously described. The gray intensity of proteins was measured using Image J software (National Institutes of Health, 9000 Rockville Pike, Bethesda, MD, USA). Primary antibodies include Anti-Nrf2 antibody (Abcam), anti-HO-1 antibody (Abcam), anti-VEGF antibody (Abcam), anti-bFGF antibody (Abcam), anti-IL-17 antibody (Abcam), anti-IL-6 antibody (Abcam), anti-TNF-α antibody (Abcam), and anti-MCP-1 antibody (Abcam). Nuclear expression of Nrf2 was detected using the NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher, No. 78833).