Chamber slides were pre-coated with poly-L-ornithine (Sigma Aldrich, USA) and 2x104 single cells were seeded per well 4 hours before the actual experiments. To staining these cells, the cells were fixed with 4% paraformaldehyde (PFA) and incubated at room temperature. Then blocking solution (5% NHS and 0.5% Triton X-100 in PBS) was added at room temperature for 1 hour. And then the cells were stained with antibodies diluted in antibody dilution buffer (1% BSA and 0.3% Triton X-100 in PBS). The following antibodies were used as primary antibodies: anti-FoxM1 (Santa cruz, USA); anti-Sox2 (R&D Systems, Minneapolis, MN); anti-Nestin conjugated to Alexa-647 (BD Biosciences, San Jose, CA, USA); anti-pH2AX (Millipore, Billerica, MA); anti-GFAP (Millipore, Billerica, MA); anti-O4 (Millipore, Billerica, MA); anti-Tuj1 (Millipore, Billerica, MA), anti-NeuN (R&D Systems, Minneapolis, MN). Analysis was performed using confocal LSM 700 (Carl Zeiss, Germany).
Anti ph2ax
Manufactured by Merck Group
Sourced in United States
Anti-pH2AX is a laboratory equipment product used for the detection and quantification of phosphorylated histone H2AX (γ-H2AX), a marker of DNA double-strand breaks. It provides a reliable and sensitive method for analyzing cellular responses to DNA damage.
Automatically generated - may contain errors
Lab products found in correlation
Anti phospho p53, by Cell Signaling Technology (2 mentions)
Anti β actin, by Merck Group (2 mentions)
Lsm 700, by Zeiss (2 mentions)
Lsm 710, by Zeiss (2 mentions)
Anti sox2, by R&D Systems (1 mentions)
Anti o4, by Merck Group (1 mentions)
Anti tuj1, by Merck Group (1 mentions)
Anti foxm1, by Santa Cruz Biotechnology (1 mentions)
Anti gfap, by Merck Group (1 mentions)
Poly l ornithine, by Merck Group (1 mentions)
Dm600 microscope, by Leica (1 mentions)
Mitoid, by Enzo Life Sciences (1 mentions)
Lsm780 confocal laser scanning microscope, by Zeiss (1 mentions)
Anti vinculin, by Merck Group (1 mentions)
Axioplan 2 microscope, by Zeiss (1 mentions)
Alexa fluor 488 anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Fluoroshield, by Merck Group (1 mentions)
Anti pgk1, by Proteintech (1 mentions)
Sta 011, by Cell Biolabs (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
8 protocols using anti ph2ax
1
Immunocytochemistry of Neural Stem Cells
2
Visualizing Mitophagy and Oxidative Stress
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
H9C2 cells were fixed with 4% PFA, permeabilized with 0.5% Triton, blocked with 3% BSA and incubated with anti‐vinculin (Sigma) or anti‐pH2A.X (Millipore) overnight at 4°C. The secondary antibody was Alexa‐Green‐488 goat anti‐mouse (Invitrogen). Images were acquired using epifluorescence microscopy (DM600 microscope; Leica). For mitophagy detection, EGFP‐LC3 plasmid and MitoID (Enzo) were used to stain autophagosomes and mitochondria, respectively. Image acquisition was performed with an LSM780 laser scanning confocal microscope (Carl Zeiss). For mitochondrial 8‐OH‐dG detection, cells were fixed with methanol for 30 min at −20°C, permeabilized with 0.2% Triton and treated with RNase A at 37°C for 1 hr, followed by denaturation with ice‐cold 25 mM NaOH in 50% ethanol, as previously described (Ohno, Oka, & Nakabeppu, 2009 ). After blocking with 10% BSA, the cells were incubated with mouse anti‐8‐OH‐dG (clone 483.15; Millipore) overnight at 4°C. The secondary antibody was goat anti‐mouse Alexa 488.
3
Immunofluorescent Detection of DNA Damage
4
Western Blot Analysis of Protein Modifications
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell lysates were prepared in RIPA buffer and Western blots were performed as previously described (67 (link)). Primary antibodies were anti-PARK7 (Santa-Cruz, clone D4, 1 : 1000), anti-PGK1 (Proteintech 17811–1-AP, 1:4,000), anti-GAPDH (Proteintech 60004–1-Ig, 1:20,000), anti–β-actin (Sigma, clone AC-15, 1:5,000), antiphospho-p53 (Cell Signaling #9284; 1:1,000), anti–P-H2AX (Millipore; 1:1,000), anti-GLO1 (1:1,000) and antimethylglyoxal (recognizing the MG-H1 modification of arginines, Cell Biolabs STA-011, 1:5,000) diluted in Tris-buffered saline (150 mM NaCl, 20 mM Tris-Cl pH 7.4, 0.05% Tween-20) containing 2% bovine serum albumin (Sigma).
5
Western Blot Analysis of DNA Damage Response
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were harvested and centrifuged at 5000 rpm for 5 min at 4 °C. The supernatant was removed, Buffer WCE 2X (100 mM TrisHCl pH 6.8, 4% SDS, 200 mM DTT) was added to resuspend the cell pellet, boiled for 10 min and then added an equal volume of SDS-PAGE Sample Loading Buffer [2X] (100 mM TrisHCl pH 6.8, 4% SDS, 200 mM DTT, 20% glycerol, 0.004% bromphenol blue) to the mixture. Cell extracts were pelleted at 15,000 g in an Eppendorf centrifuge for 15 min at 4 °C and the supernatants were analyzed by Western blotting according to [55 (link)], using the following antibodies, all diluted in TBS-T: anti-p-p53 (Ser 15; 1:1000, Santa Cell Signaling), anti-p53 (1:1000, Santa Cruz), anti-p-H2AX (Ser 139; 1:1000, Millipore), anti-SUV39H1 (44.1; 1:1000, Santa Cruz Biotechnology), anti-TDP-43 (1:5000, Proteintech), anti-H3k9me3 (1:1000, Abcam ab8898), anti-H3K9me2 (1:500, Abcam ab1220), anti-actin-HRP-conjugated (1:5000, Santa Cruz Biotechnology). These primary antibodies were detected using HRP conjugated anti-mouse and anti-rabbit IgGs and the ECL detection kit (all from GE Healthcare). Band intensities were quantified by densitometric analysis with Image Lab software (Bio-Rad).
All full length uncropped original western blots are available in the Supplementary Materials section.
All full length uncropped original western blots are available in the Supplementary Materials section.
6
Histopathological Evaluation of Tumor Samples
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Excised tumors, kidney and livers were sampled just after sacrifice and representative areas were a) formalin-fixed (24 hours) (Millipore) and paraffin-embedded and (b) snap-frozen in OCT and stored at 80ºC as previously described [32 (link)]. Tissue sections 2μM thick were stained with hematoxilin & eosin and prepared for IHC. Two experienced pathologists (MCGM and EDA) observed the samples under a Leica microscope (Leica Microsystems). IHC was performed as previously described [32 (link)] using the following primary antibodies: anti-pH2AX (Millipore); anti-cleaved PARP (cell signaling); anti-Ki67 (Millipore) and anti-BRCA2 (Millipore). TUNEL assays (Roche) were performed to detect DNA fragmentation and late apoptosis. To quantify the extent of necrosis and the IHC findings, the Dotslide analysis program (Olympus) and the Ariol Image analysis system (Olympus) were used respectively.
7
Immunofluorescence Staining Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were grown on Thermo Fisher Scientific Nunc Lab-Tek II Chamber Slides & trade, fixed with 4% paraformaldehyde for 15 min or PTEM buffer containing 20 mM PIPES (pH 6.8), 0.2%. Triton X-100, 10 mM EGTA, and 4% paraformaldehyde for 10 min, and permeabilized with 0.5% Triton X-100 for 5 min. The fixed cells were incubated for 1 h with % bovine serum albumin and then incubated overnight at 4°C with primary antibody, then the next day add the fluorophore-conjugated secondary antibody for 2 h. Samples were mounted in Vectashield Mounting Medium with DAPI (Vector laboratories, USA), and the results were observed under a confocal microscope (Zeiss LSM 710 or LSM 800, software ZEN; Zeiss, Germany). The primary antibodies were acquired from commercial sours: anti-NCAPH (Novus Biological), anti-GEN1 (Novus Biological), anti-p-H2AX (Millipore), anti-CREST (ImmunoVision, USA), anti-GFP (Invitrogen), and anti-alpha Tubulin (Abcam).
8
Western Blot Protein Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell lysates were prepared and western blots were performed using the same procedures as previously described [18 (link)]. Antibodies used were anti-PKM (Santa-Cruz, clone C-11, 1 : 1000), anti-PGP (Santa-Cruz, clone E10, 1 : 1000), anti-β-actin (Sigma, clone AC-15, 1 : 5000), anti-p53 (clone DO1, Santa-Cruz, 1 : 1000), anti-phospho-p53 (Cell Signalling, 1 : 1000), anti-P-H2AX (Millipore, 1 : 1000) and anti-tubulin (Sigma, 1 : 40 000).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!