Rabbit anti mmp 9 antibody

At 6 h and 1 day post-CNV induction, enucleated mouse eyes were fixed in 4% paraformaldehyde for 1 h at room temperature, cryoprotected in 30% sucrose at 4°C overnight, and embedded in OCT compound. Eyes were cryosectioned to 15 μm thickness. For immunofluorescence, sections were blocked in a blocking buffer (0.5% Triton, 0.2% BSA, and 5% donkey serum in PBS) for 1 h at room temperature and subsequently incubated with primary antibodies overnight at 4°C. After washing, samples were incubated with secondary antibodies for 1 h at room temperature. Goat anti-Iba1 antibody (1:250; FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan, 011-27991), rabbit anti-TMEM119 (1:500, Synaptic Systems, Göttingen, Germany, 400 002), rat anti-CD31 (1:100, BD Biosciences, NJ, USA, 553370), and rabbit anti-MMP-9 antibody (1:100; Abcam, Cambridge, MA, USA, ab38898) were used for primary antibodies, and Alexa Fluor 488-conjugated donkey anti-goat antibody (1:500; Thermo Fisher Scientific, Waltham, MA, USA, A11055), Alexa Fluor 594-conjugated donkey anti-rabbit antibody (1:500; Thermo Fisher Scientific, A21207), and Alexa Fluor 647-conjugated donkey anti-rat antibody (1:500; Jackson Immuno Research Laboratories, INC., PA, USA, 712-605-15) were used for secondary antibodies.

For western blot analysis, A549 and 95D cells were plated on culture flasks and treated with Au-NPs (control, 5 nm, 10 nm, 20 nm, and 40 nm). After 48 h, 5–10×10⁶ cells were harvested and lysed with ice-cold lysis buffer. Equal amounts of protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electrophoretically transferred onto polyvinylidene fluoride membranes. Membranes were blocked with 5% non-fat dry milk for 2 h and incubated overnight at 4°C with rabbit anti-MMP-9 antibody (1∶1000, Abcam), rabbit anti-ICAM-1 antibody (1∶500, Cell Signaling), or mouse anti-GAPDH antibody (1∶10000, Abmart). GAPDH was used as the housekeeping gene control and the expression levels of the MMP9 and ICAM-1 were normalized with respect to GAPDH. The proteins were detected with horseradish peroxidase-conjugated anti-rabbit or anti-mouse secondary antibodies and visualized with chemiluminescence reagents provided with the ECL kit (BioRad, USA). Immunoreactive bands were detected by enhanced chemiluminescence and quantified using a ChemiDoc XRS molecular imager (BioRad).

The protein expression was assessed by Western blotting. Primary antibodies, i.e., mouse anti-α-SMA antibody (SMA, Sigma-Aldrich, A2547), rabbit anti- MMP-9 antibody (Abcam, Cambridge, UK, ab76003), mouse anti-Toll-like receptor-4 (TLR-4) antibody (Abcam, ab30667), and mouse anti-β-actin antibody for internal control (Sigma-Aldrich, A5411) were reacted with the polyvinylidene fluoride (PVDF) membrane at 4 °C overnight. Subsequently, the corresponding secondary antibodies were immersed at room temperature for 1 h. The protein bands were quantified using Image J software and normalized using individual internal controls for comparison [17 (link),18 (link)].