Protein expression of proliferating cell nuclear antigen (PCNA) was studied by means of immunoblotting as previously described (Bajt et al. 2000 (link); Maes et al. 2016b (link)). In essence, liver tissue protein lysates (50 μg per lane) were resolved on 4-20% sodium dodecyl sulphate polyacrylamide gel electrophoresis under reducing conditions. Separated proteins were transferred to polyvinylidene difluoride membranes (Millipore Corporation, USA) and blocked overnight at 4°C with 5% milk in TBS/T. After washing with TBS/T, membranes were incubated with a mouse monoclonal anti-PCNA antibody (Santa Cruz Biotechnology, USA), diluted 1/2000 in blocking buffer for 2 hours at room temperature. Membranes were washed and incubated with appropriate secondary horseradish peroxidase-coupled antibody (Santa Cruz Biotechnology, USA) for 1 hour at room temperature. Proteins were visualized by enhanced chemiluminescence (Amersham Biosciences, USA) according to the manufacturer’s instructions. Densitometric analysis was performed with a GS170 Calibrated Imaging Densitometer (Bio-Rad, USA) using Quantity One 4.0.3. software (Bio-Rad, USA). For semiquantification purposes, PCNA signals were normalized against β-actin signals.
Mouse monoclonal anti pcna antibody
Manufactured by Santa Cruz Biotechnology
Sourced in United States
The Mouse monoclonal anti-PCNA antibody is a laboratory reagent produced by Santa Cruz Biotechnology. This antibody recognizes the Proliferating Cell Nuclear Antigen (PCNA) protein, which is a highly conserved nuclear protein involved in DNA replication and repair.
Automatically generated - may contain errors
Lab products found in correlation
Gs170 calibrated imaging densitometer, by Bio-Rad (2 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Enhanced chemiluminescence, by Cytiva (1 mentions)
M o m immunodetection kit, by Vector Laboratories (1 mentions)
Goat anti mouse igg alexa 488, by Thermo Fisher Scientific (1 mentions)
Lsm 710 microscope, by Zeiss (1 mentions)
β actin signal, by Merck Group (1 mentions)
Mini protean tgx precast protein gel, by Bio-Rad (1 mentions)
Iblot transfer stack, by Thermo Fisher Scientific (1 mentions)
Vectastain abc reagent, by Vector Laboratories (1 mentions)
Biotinylated anti mouse igg, by Vector Laboratories (1 mentions)
Eclipse e200 microscope, by Nikon (1 mentions)
Poly l lysine coated slides, by Agilent Technologies (1 mentions)
Streptavidin biotin peroxidase complex, by Agilent Technologies (1 mentions)
3 3 diaminobenzidine (dab), by Agilent Technologies (1 mentions)
7 protocols using mouse monoclonal anti pcna antibody
1
Quantifying Hepatic PCNA Protein Levels
2
Quantifying PCNA-Positive Cells in Liver
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Immunostaining for PCNA antigen was performed on paraffin-embedded liver tissue sections (4 μm thick) with a mouse monoclonal anti-PCNA antibody (1:50; Santa Cruz Biotechnology) and the MOM immunodetection kit (Vector, PK2002) according to the manufacturer’s instructions. The number of PCNA-positive cells were quantified from 10 fields (× 200 magnification) from 6 to 10 mice/group. No staining was observed when omitting the primary antibody.
3
Quantifying PCNA Expression in Cancer Cells
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The ES2 and OV90 cells (6 × 103 cells per 300 µL) were seeded in confocal dishes and treated with 4-MU (1 mM) or left untreated for 24 h. To visualize the expression and localization of proliferating cell nuclear antigen (PCNA), cells were incubated with a mouse monoclonal anti-PCNA antibody (1:100; catalog number: sc-56; Santa Cruz Biotechnology) for 24 h after cell fixation. The cells were then incubated with goat anti-mouse IgG Alexa 488 (1:200; catalog number: A-11001; Invitrogen, Carlsbad, CA, USA) for 1 h at room temperature. Then, cells were washed three times with 0.1% bovine serum albumin (BSA) in phosphate-buffered saline (PBS). Next, we counterstained cell nuclei with 4′,6-diamidino-2-phenylindole. Images were captured using an LSM710 microscope (Carl Zeiss, Oberkochen, Germany). Fluorescence intensity was analyzed using the ImageJ program (National Health Institutes, Bethesda, MD, USA).
4
PCNA Protein Expression Analysis
5
Immunostaining Antibodies for Cell Cycle and Apoptosis
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Antibodies were diluted in 3 % NGS from stocks prepared according to manufacturers´ instructions.
1. Mouse monoclonal anti-BrdU antibody (1:400; Cat# 11170376001, RRID:AB_514483, Roche Applied Sciences) was used to detect 5-bromo-2'-deoxyuridine, a thymidine analogue that incorporates into DNA during the S phase of the cell cycle.
2. Mouse monoclonal anti-PCNA antibody (1:500; Cat# sc-56, RRID:AB_628110, Santa Cruz Biotechnology, CA, USA). PCNA is a nuclear protein which is expressed during S-phase and G2 phase of the cell cycle.
3. Rabbit polyclonal anti-glial fibrillary acidic protein (GFAP) antibody (1:500; Cat# M0761, RRID:AB_2109952, Agilent, Santa Clara, CA, USA) was used to stain Müller glia (particularly after injury).
4. Mouse monoclonal anti-synaptic vesicle protein 2 (SV2) antibody (1:750; RRID:AB_2315387, Developmental Studies Hybridoma Bank, IA, USA) was used to stain presynaptic terminals containing vesicles in plexiform layers.
5. Rabbit polyclonal anti-PKC antibody (1:350; Cat# sc-10800, RRID:AB_2168560, Santa Cruz Biotechnology) was used to stain α, β, γ, δ and ε-subunits expressed by on-and off-BC.
6. Rabbit polyclonal anti-active caspase 3 antibody (1:350; Cat# 564096, RRID:AB_2738589, BD Biosciences, San Jose, CA, USA) was used to stain active caspase 3-dependent apoptotic cells.
1. Mouse monoclonal anti-BrdU antibody (1:400; Cat# 11170376001, RRID:AB_514483, Roche Applied Sciences) was used to detect 5-bromo-2'-deoxyuridine, a thymidine analogue that incorporates into DNA during the S phase of the cell cycle.
2. Mouse monoclonal anti-PCNA antibody (1:500; Cat# sc-56, RRID:AB_628110, Santa Cruz Biotechnology, CA, USA). PCNA is a nuclear protein which is expressed during S-phase and G2 phase of the cell cycle.
3. Rabbit polyclonal anti-glial fibrillary acidic protein (GFAP) antibody (1:500; Cat# M0761, RRID:AB_2109952, Agilent, Santa Clara, CA, USA) was used to stain Müller glia (particularly after injury).
4. Mouse monoclonal anti-synaptic vesicle protein 2 (SV2) antibody (1:750; RRID:AB_2315387, Developmental Studies Hybridoma Bank, IA, USA) was used to stain presynaptic terminals containing vesicles in plexiform layers.
5. Rabbit polyclonal anti-PKC antibody (1:350; Cat# sc-10800, RRID:AB_2168560, Santa Cruz Biotechnology) was used to stain α, β, γ, δ and ε-subunits expressed by on-and off-BC.
6. Rabbit polyclonal anti-active caspase 3 antibody (1:350; Cat# 564096, RRID:AB_2738589, BD Biosciences, San Jose, CA, USA) was used to stain active caspase 3-dependent apoptotic cells.
6
Immunohistochemical Analysis of Proliferation in Intestinal Polyps
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The middle segments of the small intestines were fixed, embedded, and sectioned using the Swiss roll technique for further immunohistochemical examination with the avidin–biotin immunoperoxidase procedure. Monoclonal mouse anti-PCNA antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) was used at a 1:200 dilution. As the secondary antibody, biotinylated anti-mouse IgG, absorbed with horse serum (Vector Laboratories, Burlingame, CA, USA) was employed at a 1:200 dilution. Staining was performed using avidin–biotin reagents (Vectastain ABC reagents; Vector Laboratories, Burlingame, CA, USA), 3,3′-diaminobenzidine and hydrogen peroxide, and the sections were counterstained with hematoxylin to facilitate orientation. All polyps were imaged at 20× magnification, and the PCNA-positive cell ratio (%) was calculated as follows: number of PCNA-stained nuclei in the polyp/number of nuclei in the polyp × 100.
7
Immunohistochemical Analysis of PCNA
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Paraffin-embedded tissue sections were mounted on poly-l -lysine-coated slides (Dako, Carpinteria, CA, USA), dewaxed in xylol, cleared in a decreasing series of alcohol, and rehydrated with distilled water. Endogenous peroxidase was blocked by treatment with H2O2 (3.7% vol) followed by H2O for 15 min. After 3 washes in 150 mM PBS, the sections underwent microwave antigen retrieval, and were exposed overnight at 4 °C to monoclonal mouse anti-PCNA antibody (1:200, Santa Cruz Biotechnology, Santa Cruz, CA, USA).
After another 3 washes in PBS, the immunocomplex was visualized with biotin-streptavidin-peroxidase complex and 3,3-diaminobenzidine (Dako, Glostrup, Denmark). The sections were lightly counter-stained with Harris hematoxylin. Negative controls were established by omitting the primary antibody and substituting immunoglobulin G for the primary antibodies.
We analyzed 10 non-consecutive sections from each immunostained kidney. The images were captured using a Nikon Eclipse E200 microscope (Amsterdam, The Netherlands) connected to a charge-coupled device (CCD) camera and ImageJ, an image-analysis software (NIH, Bethesda, MD, USA).
The tubular cell proliferation index was defined as the ratio between the nuclei expressing PCNA and the total nuclei in each tubule in every field analyzed (magnification, ×40).
After another 3 washes in PBS, the immunocomplex was visualized with biotin-streptavidin-peroxidase complex and 3,3-diaminobenzidine (Dako, Glostrup, Denmark). The sections were lightly counter-stained with Harris hematoxylin. Negative controls were established by omitting the primary antibody and substituting immunoglobulin G for the primary antibodies.
We analyzed 10 non-consecutive sections from each immunostained kidney. The images were captured using a Nikon Eclipse E200 microscope (Amsterdam, The Netherlands) connected to a charge-coupled device (CCD) camera and ImageJ, an image-analysis software (NIH, Bethesda, MD, USA).
The tubular cell proliferation index was defined as the ratio between the nuclei expressing PCNA and the total nuclei in each tubule in every field analyzed (magnification, ×40).
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