Anti timp 2

We purchased anti-Akt, anti-phospho-Akt, anti-mTOR, anti-phospho-mTOR, anti-MMP-2, anti-MMP-9, anti-GAPDH, anti-tissue inhibitor of matrix metalloproteinase (TIMP)-1, anti-TIMP-2, anti-phospho-c-Jun, and anti-phospho-p65 antibodies from Santa Cruz Biotechnology Inc (Santa Cruz, CA, USA). Surfactin and urban PM (SRM 1648a) were purchased from Sigma (St. Louis, MO, USA).

Immunohistochemical studies of TIMP-2, MMP-9, TGF-β and caspases-3 were performed on 4- to 5-μm-thick sections. After deparaffinization and blocking the endogenous peroxidases, sections were incubated with mouse anti-TIMP-2, anti-MMP-9, anti-TGF-β or anti-caspases-3 (diluted 1:200, Santa Cruz Biotechnology, Santa Cruz, CA, USA) antibodies approximately 60 min at room temperature followed by the secondary antibody (dilution 1/2000, Thermo Fisher Scientific Inc., Waltham, MA, USA) and diaminobenzidin (DAB) to develop staining. Samples were stained with Mayer’s hematoxylin for 1 min and fixed utilizing Aquatex liquid (Merck KGaA, Germany). All samples were treated keeping similar conditions having similar antibody concentration to guarantee the immunostaining would be comparable across various experimental groups.
In the immunohistochemically-stained tissues, the color intensity for the immunoreactivity of each protein was semi-quantitatively evaluated in three randomly selected fields from five different specimens. The intensity was expressed as + (weak immunoreactivity), ++ (moderate immunoreactivity), +++ (strong immunoreactivity), or ++++ (very strong immunoreactivity).

The immunolocalization technique used for TIMP-2 and Bax was performed on 3 to 4 μm thick sections according to Pedrycz and Czerny [30 (link)]. For negative controls, the primary antibody was omitted. In brief, mouse anti-Bax or anti-TIMP-2 (diluted1:200, Santa Cruz Biotechnology, Santa Cruz, CA, USA) antibodies were incubated with sections for 60 min, diluted in TBS (Tris-buffered saline)/1 % BSA (bovine serum albumin). A biotinylated secondary antibody directed against mouse immunoglobulin (Biotinylated Link Universal–DakoCytomation kit, supplied ready to use) was then added and incubated for 15 min, followed by horse radish peroxidase conjugated with streptavidin (DakoCytomation kit, supplied ready to use) for an additional 15 min of incubation. 3-amino-9-ethylcarbasole (AEC) (DakoCytomation kit, supplied ready to use) was then applied for 15 min, yielding a reddish brown color at the sites of immunolocalization of the primary antibodies. Specimens were counterstained with hematoxylin for 1 min and mounted using Aquatex fluid (Merck KGaA, Germany). The staining intensity was graded as very weak, weak, medium, or strong. All sections were incubated under the same conditions and at the same time with the same concentration of antibodies to ensure that the immunostaining would be comparable among the different experimental groups.

Western blots were carried out as previously reported [30 (link)]. The rabbit anti-Dicer (1:1000, Proteintech, USA), anti-MMP-2 (1:1000, Cell Signaling Technology, USA), anti-MMP-9 (1:1000, Cell Signaling Technology, USA), anti-TIMP-1 (1:200, Santa Cruz, USA) and anti-TIMP-2 (1:200, Santa Cruz, USA) were used for primary antibody incubation at 4°C overnight. The mouse anti-β-actin (1:1000, Cell Signaling Technology, USA) was used for the protein loading control. Each blot was repeated at least three times. The intensity of the protein bands were analyzed by densitometry after normalization to the corresponding protein controls.