Testicular tissues of rats were prepared using radioimmunoprecipitation assay buffer containing protease inhibitor. BCA (Solarbio) was used to test protein concentrations. The protein was separated by electrophoresis and transferred to membranes. The following primary antibodies were used for incubation with membranes: PI3K (1:1000; ABCAm), Akt (1:1000; Affinity Biosciences), p‐Akt (1:1000; Affinity Biosciences), caspase‐9 (1:1000; Affinity Biosciences), Bcl‐2 (1:1000; Affinity Biosciences), Bax (1:1000; Affinity Biosciences), PCNA (1:1000; Affinity Biosciences), PLZF (1:1000; Affinity Biosciences), REC8 (1:1000; Bioss), STRA8 (1:1000; Affinity Biosciences), SYCP3 (1:1000; Affinity Biosciences), GAPDH (1:1000). Then, the membranes were incubated with a secondary antibody (Bioss). Subsequent visualization with a chemiluminescent imaging system was performed (KPL).
Proliferating cell nuclear antigen (pcna)
Manufactured by Affinity Biosciences
Sourced in United States
PCNA (Proliferating Cell Nuclear Antigen) is a protein that plays a central role in DNA replication and repair processes. It acts as a processivity factor, enhancing the activity of DNA polymerases during DNA synthesis. PCNA is essential for the progression of the replication fork and the maintenance of genomic integrity.
Automatically generated - may contain errors
Lab products found in correlation
Bcl2 associated x (bax), by Affinity Biosciences (2 mentions)
Gapdh, by Affinity Biosciences (2 mentions)
Bcl 2, by Affinity Biosciences (2 mentions)
Hrp conjugated secondary antibody, by ABclonal (2 mentions)
P akt, by Affinity Biosciences (1 mentions)
Bicinchoninic acid (bca), by Solarbio (1 mentions)
Caspase 9, by Affinity Biosciences (1 mentions)
Diamidino phenyl indole dapi, by Beyotime (1 mentions)
Bx41 microscope, by Olympus (1 mentions)
Caspase 1, by Merck Group (1 mentions)
Nlrp3, by Novus Biologicals (1 mentions)
Gsdmd, by Abcam (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Bca protein assay kit, by Ipsen (1 mentions)
Tanon 5200, by Shanghai Tanon Science & Technology (1 mentions)
Vegfr2, by Abcam (1 mentions)
β actin, by ABclonal (1 mentions)
Occludin, by Abcam (1 mentions)
β actin, by Proteintech (1 mentions)
7 protocols using proliferating cell nuclear antigen (pcna)
1
Molecular Signaling in Rat Testes
2
Immunofluorescence Staining of Macrophage Markers
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Frozen sections were subjected to immunofluorescence staining using primary antibodies against CD86 (diluted 1:100; Sigma‐Aldrich), CD206 (monoclonal; diluted 1:100; Invitrogen), and PCNA (AF0239; Affinity) at 4°C overnight. On the 2nd day, the slides were washed thrice using 1× PBS for 5 min each time and incubated with the corresponding secondary antibody (diluted 1:500; Molecular Probes) for 1 h at room temperature. Diamidinophenyl indole (DAPI) (diluted 1:300; Beyotime) was used to visualize the nuclei. CD86‐ and CD206‐positive cells were detected using a confocal laser‐scanning microscope (Leica DIM8).
3
Immunofluorescence and Immunohistochemistry of Liver Samples
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Immunofluorescence and immunohistochemical examinations were conducted as described previously [3 (link)]. Briefly, frozen liver sections (4 μm) were used for immunofluorescence assays, and paraffin liver sections (4 μm) were used for immunohistochemical assays. Primary antibodies against NLRP3 (1:50, NOVUS, Centennial, CO, USA), GSDMD (1:1000, Abcam, CB, Waltham, MA, USA), Caspase-1 (1:200, Sigma, St. Louis, MO, USA), and PCNA (1:200, Affinity Biosciences, Cincinnati, OH, USA) were added and incubated overnight at 4 °C. Then, immunofluorescence images were taken by fluorescence microscopy (Zeiss-DMI8), and immunohistochemical images were obtained by an Olympus (BX41) microscope and semiquantitatively measured by Fiji software.
4
Immunohistochemical Analysis of Liver PCNA
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The formalin-fixed liver tissue was dehydrated through graded alcohols and embedded in paraffin wax (4% phosphate-buffered and formalin-fixed for 24 h at room temperature; 5 µm thick). The sections were blocked with 5% BSA for 1 h and then incubated with anti-proliferating cell nuclear antigen (PCNA; Affinity Biosciences; 1:200; cat. no. AF0239) overnight at 4°C. The slides were washed with PBS, then incubated with the HRP conjugated secondary antibody (ABclonal Biotech Co., Ltd.; 1:1,000; cat. no. AS014) for 1 h at room temperature. Staining was developed with diaminobenzidine substrate solution (DAB; Wuhan Servicebio Technology Co., Ltd.) and the sections were counterstained with hematoxylin for 1 min at room temperature, then they were dehydrated through graded alcohols, mounted and visualized under a light microscope. Data were expressed as the percentage of PCNA positive nuclei analyzed using ImageJ software (version 1.43; National Institutes of Health).
5
Liver Protein Extraction and Western Blot Analysis
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Total protein was extracted from liver tissues using RIPA lysis buffer (Beyotime Institute of Biotechnology). The protein concentration was confirmed using a BCA Protein Assay kit (Epizyme; cat. no. ZJ102) and protein samples (40 µg) were collected and subjected to SDSPAGE (6% separation gel and 10% concentration gel) and then transferred to a polyvinylidene fluoride membrane (MilliporeSigma). Next, the membranes were blocked with skimmed milk (PPLYGEN; cat. no. P1622) for 1 h at room temperature and followed by incubating with primary antibodies overnight at 4°C: native VEGF (R & D Systems; 1:1,000; cat. no. 293-VE/CF), VEGFR2 (Abcam; 1:1,000; cat. no. AB39256), PCNA (Affinity Biosciences; 1:1,000), β-actin (ABclonal Biotech Co., Ltd.; 1:1,000; cat. no. AC028) and GAPDH (Aksomics Inc.; 1:3,000; cat. no. KC-5G5) and HRP conjugated secondary antibody (ABclonal Biotech Co., Ltd.; 1:1,000; cat. no. AS014) at room temperature for 1 h. The protein band were detected ECL regents, imaged using Tanno imaging system (Tanon 5200; Tanon Science & Technology) and the protein bands analyzed with ImageJ (version 1.43; National Institutes of Health).
6
Quantitative Western Blot Analysis
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Western blot analysis was performed as described previously14 (link). Briefly, colon or cell proteins were subjected to SDS-PAGE and transferred to PVDF membranes, which were sliced for concurrent detection with different primary antibodies. β-actin (1:1000, catalog no. 66009-1-Ig, Proteintech, USA), Occludin (1:1000, catalog no. ab216327, Abcam, UK), ZO-1 (1:1000, catalog no. ab216880, Abcam, UK), PCNA (1:500, catalog no. AF0239, Affinity, USA), IL-1β (1:1000, catalog no. 12242S, CST, USA), IL-6 (1:1000, catalog no. DF6087, Affinity Biosciences, China), stat-3 (1:1000, catalog no. 12640S, CST, USA), p-stat-3 (S727) (1:1000, catalog no. ET1607-39, Huabio, China), non-phospho (Active) β-catenin (1:1000, catalog no. 8814S, CST, USA) and TNF-α (1:1000, catalog no. 41504, Signalway Antibody, USA). After incubation with the primary antibodies overnight at 4°C, the signals were detected, and protein band intensities were analyzed via densitometry in Image Lab and quantified in ImageJ software separately.
7
Puerarin Modulates P53 and Apoptosis Pathways
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Puerarin was purchased from Macklin (Art. No. P816259, China) with purity ≥ 98% (HPLC). Antibodies against P53, P21, PTEN, BRCA1, BIRC5, CTGF, Bax, Bcl-2, caspase 3, cleaved caspase 3, GAPDH, Ki67, and PCNA were purchased from Affinity Biosciences (USA); IgG H&L (Alexa Fluor® 488) was from Abcam (UK); CD3, CD4, and CD8 were from BD Biosciences Pharmingen (USA). The CCK-8 kit was obtained from MedChemExpress (USA); the cell apoptosis kit was obtained from BD Biosciences Pharmingen (USA); the EdU-594 cell proliferation detection kit was obtained from Beyotime Biotechnology (China); the TUNEL staining kit was obtained from Servicebio (China); ELISA kits for IL-6 and TNF-α were purchased from MEIMIAN (China); the ELISA kit for CA125 was purchased from ZSGB-BIO (China).
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