IHC analysis Liver tissues were fixed in 10 neutral buffered formalin solution, embedded in paraffin, and stained for routine histology. The sections were incubated at 4? overnight with primary antibody in concentrations of 1:100 and 1:200 . As a secondary antibody, horseradish peroxidase conjugated immunoglobulin G , was used for 30 min at 37?. After further washing with Tris buffered saline, sections were incubated with complex horseradish peroxidase for 30 min at 37?. Immunolocalization was performed by immersion in 0.05 3,3′ diaminobenzidine tetrahydrochloride as chromagen. Slides were counterstained with hematoxylin before dehydration and mounting. Incubation without the primary antibody was performed as a control for the background staining. Histological evaluation was performed by a pathologist who was blind to the pharmacological characteristics of the drugs. Cytoplasm proteins were isolated from 120 mg of frozen liver tissues using a Cytoplasmic Protein Extraction kit according to the protocol provided by the manufacturer.
Protein concentrations were determined using the BCA Protein Assay kit according to the protocol provided by the manufacturer . 100 L of supernatant was added to an equal volume of 2 SDS sample buffer and boiled for 5 min at 100?. The samples were then stored at 80? until analyzed. The electrophoretic mobility of the proteins analyzed in this study was determined by Nilotinib selleck SDSpolyacrylamide gel electrophoresis using 15 acrylamide concentrations. After electrophoresis, the proteins were transferred electrophoretically to a nitrocellulose filter membrane that was then blocked for 4 h in a solution of 8 nonfat dry milk in Tris buffered saline containing 0.1 tween at RT. The membrane was then incubated overnight at 4? with Smad4 antibody and GAPDH antibody which are represented on Western blotting by two distinct bands at 65 and 36 kDa. Bands were washed four times, after which they were incubated with Horseradish Peroxidase Labeled Anti Mouse IgG for 2 h and again washed four times.
The blots were developed using an ECL Western blotting kit as recommended by the manufacturer. GAPDH was probed as an internal control. GAPDH was used to confirm that an equal amount of protein was loaded in each lane. Band intensities were determined using an AlphaImager? 2200 using the SpotDenso function of AlphaEaseFC? Software Rucaparib version 3.1.2 . Statistical analysis All determinations were repeated three times, and results are expressed as the mean SD. ANOVA was used to evaluate the difference among multiple groups followed by a post hoc test for quantitative data, and RIDIT test was used for statistical analysis of qualitative data.