MLH1, MPG, FEN1, POLβ and XRCC1 expression in primary tumours was examined from formalin-fixed paraffin-embedded samples. Tissues were sectioned at 4-μm and mounted on silanized slides, which were stored at 4°C. After deparaffinization and rehydration, the sections were quenched with 3% H2O2 in methanol to block endogenous peroxidase. Bovine serum albumin at 5% (BSA) was then applied to prevent non-specific binding. The sections were incubated with anti-MPG (dilution 1:100, Abcam, mouse, EPR10959(B)), anti-Polβ (dilution 1:500, Abcam, rabbit, ab26343), anti-FEN1 (dilution 1:800, Abcam, rabbit, ab17993), anti-XRCC1 (dilution 1:50, Abcam, mouse, ab1838), anti-MLH1 (dilution 1:100, Abcam, ab92312) and PCNA (dilution: 1:400, mouse, #2586) antibodies, and then incubated with appropriate secondary antibodies (DAKO). Diaminobenzidine (DAB) was used as chromogen and the sections were counterstained with haematoxylin. Omission of the primary antibody was used as a negative control. Protein expression was evaluated using Quick Score (QS) (by assessing both staining intensity and the percentage of the stained area with a given intensity) and dichotomized in low (score between 0 and 4) and high expression (score between 5 and 12) (Supplementary Material and Methods). Only stained nuclei of malignant cells were assessed.
Ab26343
Manufactured by Abcam
Sourced in United Kingdom
Ab26343 is a laboratory equipment product. It serves a core function in scientific research and analysis. The detailed specifications and intended use of this product are not available in an unbiased and concise format.
Automatically generated - may contain errors
Lab products found in correlation
Ab17993, by Abcam (2 mentions)
Ab1838, by Abcam (2 mentions)
Ab92312, by Abcam (1 mentions)
Ab8105, by Abcam (1 mentions)
Novex precast gels, by Thermo Fisher Scientific (1 mentions)
Transblot cell apparatus, by Bio-Rad (1 mentions)
Ab135940, by Abcam (1 mentions)
Ecl western blotting detection kit, by Advansta (1 mentions)
Chemidoc xrs system, by Bio-Rad (1 mentions)
Ab16048, by Abcam (1 mentions)
Ab109132, by Abcam (1 mentions)
Ab2893, by Abcam (1 mentions)
Ht 8 oxo dg elisa kit 2, by R&D Systems (1 mentions)
Ab124741, by Abcam (1 mentions)
Ab194, by Abcam (1 mentions)
Anti rabbit igg conjugated to horseradish peroxidase, by GE Healthcare (1 mentions)
Ab7291, by Abcam (1 mentions)
Ab2352, by Abcam (1 mentions)
Anti mouse igg conjugated to horseradish peroxidase, by GE Healthcare (1 mentions)
Ecl system, by GE Healthcare (1 mentions)
7 protocols using ab26343
1
Immunohistochemical Analysis of DNA Repair Proteins
2
Protein Expression Analysis in Brain Regions
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Proteins extracts (40 μg) from striatum, motor cortex and cerebellum were separated on 4–12%-SDS polyacrylamide gels (Novex Pre-Cast gels, Invitrogene) and transferred to nitrocellulose membranes with a TransBlot cell apparatus (Bio-Rad). α-POL β (ab26343; 1:500, Abcam, Cambridge, UK), α-FEN1 (Abcam, Ab17993; 1:500), α-OGG1, (Abcam, Ab135940; 1:500), α-p53R2, (Abcam, Ab8105; 1:1000), APE1/Ref1 (sc5572; 1:100, Santa Cruz Biotechnology, USA), α-MUTYH (Abcam, Ab55551; 1:500) primary antibodies were used with α-β-Tubulin (Sigma, T5293; 1:2000) and α-LAMIN B1 (Abcam, ab16048; 1:10 000). Immunoreactions were detected with appropriate α-rabbit or α- mouse peroxidase-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, 1:10 000) and the ECL Western blotting detection Kit (Advansta). Signals were captured with ChemiDoc XRS system (Biorad) and quantified with Image Lab software.
3
Antibody-Based Assays for DNA Damage
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Antibodies used in this experiment are listed here: HT 8-oxo-dG ELISA Kit II(R&D Systems China Co, Ltd.No.4380–192-K), APE1(ab189474, Abcam), FEN1(ab109132, Abcam), Pol Beta (ab26343, Abcam) and XRCC1(ab1838, Abcam) antibodies, anti-γ-H2AX antibody (ab2893, Abcam).
4
Western Blot Analysis of DNA Repair Proteins
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Total cell extracts were prepared from exponentially growing cells and were subjected to electrophoresis in 10% SDS-polyacrylamide gel and then transferred onto a PVDF membrane. The membrane was blocked with 5% skim milk. To detect XPA, APE1, or α-tubulin, the membrane was incubated in 1:100 dilution of anti-XPA monoclonal antibody (ab2352, Abcam), 1:2000 dilution of anti-APE1 monoclonal antibody (ab194, Abcam), or 1:10000 dilution of anti-α-tubulin monoclonal antibody (ab7291, Abcam) overnight. After washing with phosphate-buffered saline containing 0.05% Tween 20, the membrane was incubated with 1:2500 dilution of anti-mouse IgG conjugated to horseradish peroxidase (GE Healthcare Bio-Sciences). To detect OGG1 or DNA polymerase β (Pol β), the membrane was incubated with 1:10000 dilution of anti-OGG1 monoclonal antibody (ab124741, Abcam) or 1:1000 dilution of anti-Pol β polyclonal antibody (ab26343, Abcam) overnight. After washing with phosphate-buffered saline containing 0.05% Tween 20, the membrane was incubated with 1:2500 dilution of anti-rabbit IgG conjugated to horseradish peroxidase (GE Healthcare Bio-Sciences). The proteins were visualized by chemiluminescence using the ECL system (GE Healthcare Bio-Sciences).
5
ELISA-based Pol β DNA Binding Assay
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ELISA-based affinity assays was used to assay WT or R152C Pol β and DNA binding. First, a 96-well ELISA plate was coated with streptavidin (1 μg/well) and incubated overnight (4°C). Then, biotin-labeled Pol-GAP DNA substrate (1 pmol/well) was immobilized onto a 96-well plate (overnight, 4°C) and washed 3 times with PBS, followed by the addition of 0-1 ug WT or R152C Pol β recombinant proteins. Binding of Pol β was detected using a rabbit anti-Pol β antibody (Abcam, ab26343) and goat anti-rabbit secondary antibody-conjugated HRP (SC-2004). The color was developed by adding tetramethylbenzidine (TMB) and stopped by addition of 1 M H2SO4. The OD450 value was read by a microplate reader.
6
Ultrastructural Localization of DNA Polymerase β in Hippocampal Neurons
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Hippocampus from Tg and Wt mice (n = 3 for each category) was fixed with 0.1% glutaraldehyde and 4% paraformaldehyde in 0.1 M Phosphate buffer for 24 h at 4 °C. Tissues were then rinsed in 0.1 M phosphate-buffer, dehydrated in ethanol (30 and 50%, 10 min each) at 4 °C, then 70 and 96% 10 min each at − 20 °C, and embedded in LRWhite resin (3 × 1 h − 20 °C, then overnight at 4 °C), and polymerized under UV for 3 days at 4 °C. Ultrathin sections (85 nm) were made with a ultracut (Leica EM UC7). After 1 h saturation with 0.1 M Tris, 150 mM NaCl in 1% BSA and 1% NGS1, immunolabeling was carried out using Polβ antibody (ab26343, Abcam). Polβ labeling was revealed with a goat anti-rabbit IgG gold conjugate (18 nm in diameter) (Jackson ImmunoResearch Laboratories). Staining of the ultrathin sections was carried out using 2% uranyl acetate in 50% ethanol. Labeling was observed under a Zeiss EM 900 electron microscope and acquisition realized with camera gatan (Orius SC 1000). Three sections per hippocampus, at the CA1 level, were observed with Zeiss EM 900 microscope and acquisition (100–200 images per section) realized with camera gatan (Orius SC 1000).
7
Immunohistochemical Analysis of DNA Polymerase β
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IHC was done centrally on single slides at the Fondazione Filarete, as previously reported [11 (link)]. Sections were immune-stained with anti-DNA polymerase β antibody ab26343 (Abcam, Cambridge, UK), and incubated with biotinylated secondary goat anti-rabbit antibody (VC-BA-1000-MM15, Vector Laboratories, Burlingame, CA, USA). Sections were labeled by the avidin–biotin–peroxidase (ABC) procedure with a commercial immunoperoxidase kit (VECTASTAIN Elite ABC-Peroxidase Kit Standard, VC-PK-6100-KI01, Vector Laboratories, Burlingame, CA, USA). The immune reaction was visualized with 3,3′-diaminobenzidine peroxidase DAB substrate kit (VC-SK-4100-KI01, Vector Laboratories, Burlingame, CA, USA) substrate and sections were counterstained with Mayer’s hematoxylin. Figure S1 shows representative images of negative and positive DNA polymerase β staining.
A semiquantitative H-score (percentage of positive tumoral cells x intensity: 0 = negative, 1 = slight, 2 = moderate, 3 = strong) was calculated independently by two pathologists. In case of disagreement, a third opinion was requested.
A semiquantitative H-score (percentage of positive tumoral cells x intensity: 0 = negative, 1 = slight, 2 = moderate, 3 = strong) was calculated independently by two pathologists. In case of disagreement, a third opinion was requested.
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