Mmp 9 ab

Cells were lysed in PBS containing 1% nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 5 μg/mL aprotinin, 100 μg/mL phenylmethylsulfonyl fluoride, 1 μg/mL pepstatin A, and 1 mM ethylenediaminetetraacetic acid (EDTA) at 4°C for 20 minutes. Total lysates were quantified using a microBCA kit (Thermo Fisher Scientific, Waltham, MA). Proteins (10 μg) were resolved by SDS-polyacrylamide gel electrophoresis and electrophoretically transferred to a PVDF membrane. The membrane was blocked in 5% fat-free milk in PBST buffer (PBS with 0.05% Tween-20) followed by incubation overnight with the following primary antibodies diluted in PBST buffer: TM antibody (Ab), MMP-2 Ab, JNK Ab, pJNK Ab, p38 Ab, pp38 Ab, c-jun Ab, c-fos Ab, lamin B1 Ab (diluted used in 1 : 1,000, all from Santa Cruz Biotech [Dallas, TX]), MMP-9 Ab (Abcam, Cambridge, UK), p65 Ab, and p53 Ab (Genetex, Irvine, CA). The primary antibodies were removed, and the membrane was washed extensively in PBST buffer. Subsequent incubation with horseradish peroxidase-conjugated goat anti-rabbit antibodies (1 : 20,000, Santa Cruz Biotech) was performed at room temperature for 2 hours. The membrane was washed extensively in PBST buffer to remove any excess secondary antibodies, and the blot was visualized with enhanced chemiluminescence reagent (GE Healthcare).

Slides from paraffin-embedded lung tissues were deparaffinized, and Sudan Black was used to reduce autofluorescence. Tris-EDTA buffer was used for Ag retrieval, and permeabilization was achieved by using 1% Triton X. Following blocking with 2% BSA in TBST (TBS + 0.1% Tween 20), tissues on slides were incubated with MMP9 Ab (Abcam, host rabbit) in 1% BSA in TBST overnight at 4°C. After washing, the tissues were incubated with anti-rabbit 488 Ab in 1% BSA in PBS for 1 h at room temperature, followed by washing and counterstaining with DAPI. Ultimately, coverslips were mounted with use of mounting medium. Images were taken with a DeltaVision microscope (20× objective, XY 2048 × 2048) using SoftWoRx software. Analysis was performed with ImageJ with the use of three standardized macros (Supplemental Macro Codes 1–3; https://doi.org/10.6084/m9.figshare.21930690.v1).