Evaluation. Immunohistochemical evaluation was performed as previously described [25]. Briefly, paraffin-embedded renal
Evaluation. Immunohistochemical analysis was performed as previously described [25]. Briefly, paraffin-embedded renal tissue sections were dewaxed with xylene, dehydrated with a gradient PKCβ Modulator Purity & Documentation series of alcohol, incubated with H2O2, and sealed with goat serum. Subsequently, sections have been incubated with main and secondary antibodies and labeled with horseradish enzyme. DAB was utilized for colour development. Lastly, all sections had been observed and photographed below a DP73 microscope (Olympus, Tokyo, Japan). two.eight. TUNEL Assay. Paraffin-embedded renal tissue sections were pretreated in accordance with the TUNEL apoptosis detection kit (Roche, Basel, Switzerland) manufacturer’s directions then wetted for 60 min with 50 L of TdT enzyme reaction option at 37 . Just after 30 min reaction with antifluorescent antibody within the dark, sections have been incubated with DAB (5000 L) working remedy for 50 min at area temperature. All sections have been captured employing a fluorescence inverted microscope (TE2000, Nikon). Apoptosis rates were calculated in six noncontinuous fields of every section by ImageJ software program. 2.9. Determination of Protein Expression. Protein expression levels of Bax, Bcl-2, and cleaved caspase 3 (Wanlei Biotechnology, Shenyang, China) in renal tissues have been determined by western blot analysis. Briefly, frozen kidney tissues have been lysed with radioimmunoprecipitation assay lysis buffer mixed with phenylmethylsulfonyl fluoride (Beyotime Biotechnology, Shanghai, China). Just after detection of total protein concentrations using a bicinchoninic acid assay kit (Beyotime Biotechnology), samples with equal volumes of protein were separated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes, which were incubated with main antibodies of Bax (1 : 1000), Bcl-2 (1 : 500), andTable 1: The catalog numbers of all kits. Kit name Malondialdehyde Hydrogen peroxide Superoxide dismutase Glutathione Myeloperoxidase Interleukin-6 Interleukin-1 20-Hydroxystilbenetetraenoic acid Prostaglandin E2 Leukotriene B4 Phospholipase A2 Abbreviations MDA H2O2 SOD GSH MPO IL-6 IL-1 20-HETE PGE2 LTB4 PLA2 Catalog number A003-1-2 A064-1-1 A001-3-2 A006-2-1 A044-1-1 H007-1-2 H002-1-2 JL48233 H099-1 H552-1 H243-cleaved caspase three (1 : 1000) in Key Antibody Dilution Buffer (Leagene Biotechnology, MMP-1 Inhibitor manufacturer Beijing, China) overnight at four . Soon after washing, membranes were incubated with goat anti-rabbit secondary antibody (ZSGB-BIO, Beijing, China) at 37 for 2 h. All protein bands had been captured with Amersham Imager 600 software (GE, Boston, MA, USA) and quantified with ImageJ. 2.ten. Determination of Gene Level. Gene expression levels of cytochrome P450 (CYP) 4A1, CYP4A2, CYP4A3, CYP4A8, cyclooxygenase 1 (COX1), cyclooxygenase two (COX2), leukotriene B4 receptor 1 (BLT1), calcium-independent phospholipase A2 (iPLA2), secreted phospholipase A2 (sPLA2), and cytosolic phospholipase A2 (cPLA2) in renal tissues were determined with real-time PCR analysis, as previously described [26]. All primers (Table 2) have been synthesized by Shanghai Bioengineering Co. (Shanghai, China). GAPDH mRNA expression levels had been applied as a reference to quantify relative expression levels of genes. Gene levels have been quantified based on the 2-Ct process. 2.11. Statistical Analysis. All data represent the imply SEM and were analyzed making use of IBM SPSS Statistics 23 software (Armonk, NY, USA). Statistical evaluation was performed by means of one-way ANOVA, followed by Tukey’s post hoc test. Mea.
