Anti pcna antibody

Tissue samples were fixed with 4% formalin and embedded in paraffin. The slices were cut to 5 μm thickness. IHC was performed by blocking the rehydrated tissue sections using bovine serum overnight at 4°C, and subsequently applying the Anti-Ki67 antibody (Abcam, Cat. no. ab15580) and Anti-PCNA antibody (Abcam, Cat. no. ab92729). The sections were washed and then subjected to incubation with biotinylated anti-mouse IgG or biotinylated anti-rabbit IgG (Vector Laboratories, CA, USA). The ABC method (Vector Laboratories, CA, USA) was employed along with 3,3’- diaminobenzidine (Dojindo Laboratories, Kumamoto, Japan) as a substrate to detect the staining. The sections were visualized using an AX-80 microscope (Olympus, Tokyo, Japan), and Image J software (http://imagej.nih.gov/ij/) was employed for image analysis and quantification of positive expression. Statistical analysis was conducted using One-Way ANOVA.

To assess the morphological integrity, the harvested tissues at 24 hours after reperfusion were fixed in paraffin, sectioned and analysed by haematoxylin and eosin staining (H&E). Histological score of the kidney (HSK) was evaluated in a blind manner by two experienced pathologists. As described previously,² three fields per section were scored on a scale of 0‐4 (0, 0%; 1, 0%‐5%; 2, 5%‐25%; 3, 25%‐75%; and 4, 75%‐100%). The effect of LESW on apoptosis, cell proliferation and small vascular density was observed at 3 days after reperfusion. Apoptosis was evaluated with terminal transferase–mediated deoxyuridine triphosphate nick‐end‐labelling (TUNEL) assay (Roche) according to the manufacturer's protocol. Proliferation of renal cell was detected by using antiproliferating cell nuclear antigen (anti‐PCNA) antibody (Abcam). Small vascular density was measured by anti‐CD34 antibody (Abcam). Anti‐SDF‐1 antibody (Abcam) was used to observe the cell types that express SDF‐1 at 24 hours after reperfusion. Immunohistochemical assays for the staining of anti‐PCNA, anti‐CD34 and anti‐SDF‐1 were performed according to our previous protocol.²³

The ApopTag Plus Fluorescein In Situ Apoptosis Detection Kit (EMD Millipore, Bedford, MA, USA) was used as per manufacturer's instructions to quantify the number of apoptotic cells in the tibial GP of 7-week-old mice. Proliferating cell nuclear antigen (PCNA) immunohistochemistry (IHC) was performed using a 1:4000 dilution of an anti-PCNA antibody (Abcam, Cambridge, UK) followed by the Vectastain elite ABC rabbit kit (Vector Laboratories, Peterborough, UK) on paraffin-embedded sections from 3-week-old mice (when rates of proliferation were likely to be highest). Sections were counterstained by Haematoxylin and viewed using a Zeiss AxioImager brightfield microscope. The Haematoxylin-DAB colour deconvolution plugin was used in Fiji to calculate the percentage of PCNA+ve cells in the proliferating zone of the GP of each sample.

Sinoporphyrin sodium (DVDMS) was provided by Jiangxi Qinglong Group Co. Ltd (Jiangxi, China). Dipalmitoylphosphatidylcholine (DPPC) and 1, 2‐Distearoyl‐sn‐glycero‐phosphoethanolamine‐N‐[met‐hoxy (poly‐ethyleneglycol)‐2000] (DSPE‐PEG2000) were purchased from Avanti Polar Lipids Inc (Alabaster, AL, USA). Cholesterol, 2′,7′‐dichlorofluorescin diacetate (DCFH‐DA) and 4′,6‐diamidino‐2‐phenylindole (DAPI) were purchased from Sigma‐Aldrich (St. Louis, MO, USA). Fetal bovine serum (FBS), high glucose DMEM, trypsin‐EDTA and penicillin‐streptomycin were purchased from Gibco Life Technologies (Aargau, Switzerland). Cell Counting Kit‐8 (CCK‐8) was purchased from Dojindo Laboratories (Tokyo, Japan). Calcein‐AM and Propidium iodide (PI) were purchased from Invitrogen (Eugene, Oregon, USA). TdT‐mediated dUTP nick end labelling (TUNEL) kit was purchased from Roche Company (Shanghai, China). Anti‐PCNA antibody was purchased from Abcam (Cambridge, UK). Anti‐Ki‐67 antibody was purchased from Santa Cruz Biotechnology Inc (California, USA). The U87 human glioma cell line was purchased from the American Type Culture Collection. Female BALB/c athymic nude mice were obtained from Beijing Weitonglihua Experimental Animal Technology Co. Ltd (Beijing, China).

Staining of PCNA was performed at Biopathology Institute Corporation (Oita, Japan) using rabbit polyclonal anti-PCNA antibody (Abcam, Tokyo, Japan) and biotin-labeled anti-rabbit IgG antibody according to standard methods.

Protein samples were separated by 8%–12% SDS-PAGE and transferred to nitrocellulose membranes (Millipore). After blocking with 5% milk in TBST for 1h, the membranes were incubated overnight at 4°C with primary antibodies. Subsequently, membranes were washed and incubated with secondary antibodies (1:10,000) and detected using the chemiluminescence method. The intensity of the bands was quantified by ImageJ software. Antibodies included anti-NLRP3 antibody (1:1,000, CST), anti-GSDMD antibody (1:1,000, Abcam), anti-N-GSDMD antibody (1:1,000, Abcam), anti-Capspase-1 p20 antibody (1:1,000, AdipoGen), anti-4-HNE antibody (1:1,000, Abcam), anti-HDAC3 antibody (1:1,000, Proteintech), anti-HADHA antibody (1:1,000, Abcam), anti-Ace-lys antibody (1:1,000, Abcam), anti-H3 antibody (1:1,000, Proteintech), anti-COX4 antibody (1:1,000, Abcam), anti-PCNA antibody (1:1,000, Abcam). Anti-GAPDH (1:5,000, Invitrogen) was used as an internal control.
For acetylation-immunoprecipitation, cells were lysed with immunoprecipitation buffer [supplemented with TSA (10 mM)] and sonicated. The samples were immunoprecipitated with protein A/G beads (Sigma) overnight at 4°C, washed three times in lysis buffer, resolved by loading buffer, and analyzed by Western blotting.

Paraffinized tissue sections (2.5 μm-thick) from experimental mice were stained with the following antibodies: anti-α-SMA (1:1000, SMA clone 1A4, DAKO), anti-PCNA (1:800, anti-PCNA antibody, Abcam) and anti-MMP-9 (1:400, MMP-9, Santa Cruz Biotechnology, Inc.). In each staining group, isotype controls without the primary antibody were included and exhibited no staining. In addition, αSMA and PCNA quantification was performed by counting positively stained cells in the intima and was expressed as the number of positive cells/high power field (HFP) in the intima. For the quantification of MMP-9, histological software (NIS-Elements AR ver5. 11.00) was used to measure the intimal MMP-9-positive area (μm²)/intima.