Anti pcna

Immunohistochemistry was carried out using formalin-fixed paraffin-embedded (FFPE) sections as described [28 (link)]. The sections were blocked in phosphate buffered saline-tween 20 (PBST) using 10% normal goat serum. The primary antibodies were diluted in PBST containing 5% goat serum. The primary antibodies used were: anti-AFP (Santa Cruz; rabbit polyclonal; 1:50); anti-CD31 (Dako; mouse monoclonal; 1:50); anti-PCNA (Cell Signaling; mouse monoclonal; 1:100); anti-OPN (Millipore; rabbit polyclonal; 1:500); anti-CyclinB1 (1:100, rabbit polyclonal; Cell Signaling). Secondary antibodies were diluted in PBST containing corresponding 2.5% blocking serum. The signals were developed by avidin-biotin-peroxidase complexes with a DAB substrate solution (Vector laboratories). Images were analyzed using an Olympus microscope.

Western blotting was performed as previously described [16 (link)]. Briefly, cell lysates were prepared from parental wild type or miR-214KO PC3 and MDA-PCa-2b cells using lysis buffer (Cell Signaling Technology, Danvers, MA, USA) containing a protease inhibitor cocktail (Roche, Indianapolis, IN, USA). Protein concentrations were determined using the Bio-Rad protein assay reagent (Bio-Rad, Hercules, CA, USA). Cell lysates (40 μg) were separated and transferred to a polyvinylidene difluoride membrane (Millipore, Billerica, MA, USA). Membranes were incubated overnight at 4 °C with anti-PCNA, VEGFR2, E-Cadherin, N-Cadherin, Vimentin, PTK6, and GAPDH (Cell Signaling Technology), CD31 (Life Technologies), VEGFA (Abcam), CXCR4, SESN3, PD-L1, ALK, and SAA1 (Abclonal Technology, and the appropriate HRP-conjugated secondary antibodies for 1 h at room temperature. All primary antibodies were used at a concentration of 1:1000. Following the incubation, membranes were washed, and protein bands were detected with Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific, Waltham, MA, USA).

The following primary antibodies were used: anti-Akt (monoclonal rabbit, C67E7, #4691), anti-phospho (p)-Akt (S473, monoclonal rabbit, D9E, #4060), anti-Bcl-xL (monoclonal rabbit, 54H6, #2764), anti-Bcl-2 (polyclonal rabbit, #2876), anti-Cyclin D1 (monoclonal rabbit, 92G2, #2978), anti-CREB (monoclonal rabbit, 48H2, #9197), anti-p-CREB (S133, monoclonal rabbit, 87G3, #9198), anti-GAPDH (monoclonal rabbit, D16H11, #5174), anti-IGF-IR (monoclonal rabbit, D23H3, #9750 and monoclonal rabbit, D4O6W, #14,534), anti-phospho (p)-IGF-IR (Y1135/1136, monoclonal rabbit, 19H7, #3024), anti-PCNA (monoclonal rabbit, D3H8P, #13,110) and anti-Rb (polyclonal rabbit, #9313) (all purchased from Cell Signaling Technology). The SS18-SSX fusion protein was visualized using an anti-SS18/SYT antibody (#8819 Santa Cruz Biotechnology) detecting the N-terminus of SS18 (which is retained in the SS18-SSX fusion oncoprotein). Secondary antibody labeling (Bio-Rad) and immunoblot development were performed using an enhanced chemiluminescence detection kit (SignalFire ECL Reagent; Cell Signaling Technology) and a Molecular Imager ChemiDoc system (Image Lab Software; Bio-Rad), as described previously [14 (link), 40 (link)].

Mounted sections were submerged in sodium citrate buffer (0.1 M, pH 6) and left in a water bath for 15 min to expose antigen epitopes. This was followed by the incubation of the sections with 0.3% H₂O₂ in methanol to prevent any nonspecific binding to endogenous peroxidase. Rabbit anti-rat primary antibodies were added to the sections and incubated overnight at 4 °C. Rabbit monoclonal antibodies against Bax [E63] (ab32503), β-catenin [E247] (ab32572,) COX2 [EPR12012] (ab179800), Ki67 antibody (ab15580), and NF-κB-p65 (ab16502) were purchased from Abcam (Cambridge, CB2 0AX, UK), while anti-PCNA and anti-caspase-3 were purchased from Cell Signaling Technology Inc. (Danvers, MA, USA). Next, the slides were rinsed with PBS and subjected to incubation with the secondary antibody polyvalent biotinylated goat-anti-rabbit for 10 min at room temperature (1:200 dilution). The standard staining protocol was conducted using the Universal LSAB kit and DAB plus substrate kit. Additional counter-staining was carried out using hematoxylin. The slides were examined and photographed under an Olympus DP71 optical microscope. Ten fields were then chosen at random for the quantification of positive cells in individual samples (×400).

Western blot assays were performed as previously described [29 (link)]. The primary antibodies used in this study were anti-SOX9 (#A2479, ABclonal, Wuhan, China; 1:2000), anti-PCNA (#13110, Cell Signaling Technology, Shanghai, China; 1:1000), anti-Ki67 (#ab16667, Abcam, Shanghai, China; 1:1000), anti-CDK2 (#ab232753, Abcam, Shanghai, China; 1:1000), anti-CYP11A1 (#D122183, Sangon, Shanghai, China; 1:1000), anti-CYP19A1 (#A2161, ABclonal, Wuhan, China; 1:1000), anti-StAR (#8449S, Cell Signaling Technology, Shanghai, China; 1:1000), anti-caspase3 (#19677-1-AP, Proteintech, Wuhan, China; 1:1000), anti-GAPDH (#TA802519, ORIGENE, Wuxi, China; 1:3000), and anti-β-tubulin (#AC008, ABclonal, Wuhan, China; 1:3000). The corresponding HRP-conjugated secondary antibodies obtained from Sangon Biotech (Shanghai, China) were diluted in 0.25% BSA/TBST solution. The protein levels of β-tubulin and GAPDH served as internal controls in oxidative and non-oxidative experiments, respectively. Each group had three independent biological replicates. The replicates with the highest representativeness were selected and are shown in the figures, and their corresponding normalized fold change values were calculated and are listed under the blot images.