Human colonic tissue sections were analyzed by imunofluorescence as we previously described [2 (link)]. Briefly we used anti-ALDH1 (BD Pharmingen, Franklin Lakes, 1:50), anti-RXR-alpha (Santa Cruz, 1:50), anti-RAR-alpha (Santa Cruz, 1:50) and anti-MCM2 (Abcam, Cambridge 1:100) as primary antibodies. The use of human tissues was approved by Institutional Review Boards of Thomas Jefferson University and the Christiana Care Health Services, Inc (FWA00006557).
Anti mcm2
Manufactured by Abcam
Sourced in United Kingdom
Anti-MCM2 is a primary antibody that specifically targets the MCM2 (Minichromosome Maintenance Complex Component 2) protein. MCM2 is a key component of the MCM complex, which plays a crucial role in the initiation and regulation of DNA replication in eukaryotic cells. This antibody can be used to detect and quantify the expression of MCM2 in various sample types.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (3 mentions)
Phospho histone h3 ser10, by Merck Group (2 mentions)
Picrosirius red, by Abcam (2 mentions)
Prolong diamond antifade mountant, by Thermo Fisher Scientific (2 mentions)
Anti claspin, by Thermo Fisher Scientific (2 mentions)
Anti orc2, by Thermo Fisher Scientific (2 mentions)
Anti atr, by Proteintech (2 mentions)
F4 80, by Abcam (2 mentions)
Anti ki67, by Abcam (2 mentions)
Anti actin, by Merck Group (2 mentions)
Goat anti mouse hrp, by Merck Group (2 mentions)
Goat anti rabbit hrp, by Cell Signaling Technology (2 mentions)
Crescendo ecl substrate, by Merck Group (2 mentions)
Anti mcm4, by Cell Signaling Technology (2 mentions)
Anti smad2 3, by Cell Signaling Technology (2 mentions)
Anti mcm2, by Cell Signaling Technology (2 mentions)
Anti p21, by Santa Cruz Biotechnology (2 mentions)
Anti androgen receptor, by Abcam (2 mentions)
Anti aldh1, by BD (1 mentions)
Ez magna rip kit, by Merck Group (1 mentions)
13 protocols using anti mcm2
1
Immunofluorescence Analysis of Colon Tissue
2
RNA-ChIP for Transcription Factors
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RNA-ChIP was performed using the EZ-Magna RIP kit (Millipore) per the manufacturer's instructions as previously described. In brief, 107 cells were lysed with RIP lysis buffer with one freeze-thaw cycle. Cell extracts were coimmunoprecipitated with anti-TCEA1, anti-RBMX, anti-MCM2 or anti-CBX3 (Abcam), and the retrieved RNA was subjected to real-time qPCR analysis. Normal rabbit IgG was used as a negative control.
3
Immunohistochemical Analysis of Cell Proliferation and Apoptosis
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After heat-induced antigen retrieval in boiling sodium citrate buffer for 20 minutes, the rehydrated sections were incubated in phosphate buffer saline (PBS) with 10% goat normal serum and 1% bovine serum albumin for blocking. The sections were then incubated in anti-PCNA (Merck, Darmstadt, Germany), anti-MCM2 (Abcam, Cambridge, UK), or anticleaved caspase-3 (Cell Signaling, Danvers, USA) rabbit polyclonal antibody overnight at 4°C. On the second day, after incubation in biotinylated goat anti-rabbit antibody (Abcam, Cambridge, UK) for 1 hour and streptavidin-horseradish peroxidase for 1 hour, 3, 3′-diaminobenzidine (DAB) substrate was added to sections. The reaction was stopped once the appropriate color was developed and the sections were counterstained in hematoxylin for 30 seconds, dehydrated, cleared in xylene, and mounted in DPX. The numbers of positive cells were quantified by counting positive cells in a 700 μm2 field (100X). Three mice in each group and 4 fields from each mouse were acquired.
4
Western Blot Analysis of DNA Repair Proteins
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Cells were harvested and lysed in RIPA buffer containing protease inhibitor cocktail (Roche, Switzerland). The 20 μg of total protein were separated by SDS-PAGE and then transferred to PVDF membrane. After blocking in 10% non-fat milk for 1 h at room temperature, membranes were incubated with primary antibodies overnight at 4° C. The membranes were then incubated with horseradish peroxidase-labeled secondary antibodies, and visualized with ECL. The primary antibodies used were anti-Blm (1:1000, Invitrogen, USA), anti-Wrn (1:1000, Invitrogen), anti-Recql4 (1:1000, Invitrogen), anti-Timeless (1:1000, Invitrogen), anti-Ddx11 (1:1000, Invitrogen), anti-Pot1 (1:1000, Invitrogen), anti-Fancd2 (1:1000, Novus Biologicals, USA), anti-Terf1 (1:1000, Invitrogen), anti-Gins2 (1:500, Santa Cruz, USA), anti-Top2a (1:1000, Novus Biologicals), anti-Mcm7 (1:1000, Santa Cruz), anti-Mcm2 (1:1000, Abcam, UK), anti-PCNA (1:1000, Abcam), anti-H2B (1:1000, Abcam), anti-tubulin (1:5000, Proteintech, USA), anti-actin (1:1000, Santa Cruz).
5
Picrosirius Red and Immunofluorescence Staining
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Picrosirius red (Abcam; Cat# ab245887) and immunofluorescence staining were performed by standard methods using 8 μm paraffin sections. Antibodies used for immunofluorescence staining were F4/80 (Abcam; Cat# ab6640; 1:100), Ly6g (Abcam; Cat# ab25377; 1:100), phospho-histone H3 Ser10 (Millipore; Cat# 06-570; 1:200), anti-Claspin (Thermofisher; Cat# PA5-102840; 1:100), anti-MCM2 (Abcam; Cat# ab4461; 1:100), anti-Orc2 (Thermofisher; (Cat# PA5-70227; 1:100), and anti-ATR (Proteintech; Cat# 19787-1-AP; 1:100).
Sections were blocked in 3% BSA-PBST, and antibodies were diluted using 1% BSA-PBST. Primary antibodies were incubated overnight at 4 °C. Secondary antibodies were incubated for 1 h at room temperature. Slides were stained with DAPI during washing and coverslips mounted with ProLong Diamond Antifade Mountant (Invitrogen; Cat# P36961).
Sections were blocked in 3% BSA-PBST, and antibodies were diluted using 1% BSA-PBST. Primary antibodies were incubated overnight at 4 °C. Secondary antibodies were incubated for 1 h at room temperature. Slides were stained with DAPI during washing and coverslips mounted with ProLong Diamond Antifade Mountant (Invitrogen; Cat# P36961).
6
Picrosirius Red and Immunofluorescence Staining
7
Antibodies for Myogenic Lineage Analysis
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Antibodies used in this study are: anti-PAX7 (DSHB, deposited by Kawakami, A.), anti-MF20 (DSHB, deposited by Fischman, D.A.), anti-Akt (Proteintech, 10176-2-AP), anti-phosphorylated-Akt (CST, 4060), anti-eIF4E (ZENBIO, 384193), anti-phosphorylated-eIF4E (CST, 9741), anti-eIF4EBP1 (ZENBIO, R24197), anti-phosphorylated-eIF4EBP1 (CST, 2855), anti-MyoD (Santa cruz, sc-32758X), anti-MyoD(Active motif, 39991), anti-Mcm2(Abcam, ab4461), anti-Myog (Santa cruz, sc-12732), anti-ACTB (Abclonal, AC026), anti-FLAG (Abclonal, AE005), anti-mouse secondary antibody conjugated with Alexa Fluor 488 (Invitrogen, A-21202), anti-mouse secondary antibody conjugated with Alexa Fluor 555 (Invitrogen, A-31570), anti-rabbit secondary antibody conjugated with Alexa Fluor 488 (Invitrogen, A-21206), anti-rabbit secondary antibody conjugated with Alexa Fluor 555 (Invitrogen, A-31572), HRP anti-mouse secondary antibody (beyotime, A0216), HRP anti-rabbit secondary antibody (beyotime, A0208).
8
Protein Expression Analysis in Cell Lysates
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After being washed with cold phosphate-buffered saline (PBS), cells were lysed in radioimmunoprecipitation assay (RIPA) buffer (Biotechnology). The collected lysates were centrifuged at 12,000 ×g at 4 °C for 10 minutes, and then the total protein was quantified using a commercial bicinchoninic acid (BCA) kit (Biotechnology). Then, 30 µg of protein was loaded into each lane, and the protein was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; 8–12%), and transferred to a polyvinylidene difluoride (PVDF) membrane (EMD Millipore). Then, the membrane was blocked with skimmed milk and incubated overnight with the following antibodies: anti-TPD52 (1:10,000; Abcam), anti-Ki-67 (1:5,000; Abcam), anti-MCM2 (1:1,000; Abcam), anti-proliferating cell nuclear antigen (1:1,000; Cell Signaling Technology), and anti-GAPDH (1:10,000; Proteintech Group). After that, the membrane was washed 3 times with 1% tris-buffered saline (TBS) and the following secondary antibodies were added: horse-radish peroxidase (HRP) conjugated Goat Anti-Mouse IgG (1:5,000; Wuhan Boster Biological Technology) and HRP-conjugated Goat Anti-Rabbit IgG (1:5,000; Wuhan Boster Biological Technology). The protein bands were detected with a BeyoECL Plus kit (Biotechnology) and quantified by ImageJ.
9
Immunohistochemical Analysis of Xenograft Tumors
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The xenograft tumors were fixed in 2% paraformaldehyde for 24 hours. The paraffin-embedded tissue was cut into 5-µm sections. The sections were deparaffinized, rehydrated, and then subjected to high pressure for antigen retrieval (pressure, 15PSI; temperature, 121 °C) in EDTA antigen retrieval buffer (1 mM EDTA; pH 8.0). Endogenous peroxidase was inactivated with 3% hydrogen peroxide, and 1% bovine serum albumin (Sigma) was used to block non-specific binding for 1 hour. Anti-Ki-67 (1:200; Abcam), anti-MCM2 (1:200; Abcam), and anti-PCNA (1:1,000; Cell Signaling Technology) antibodies were used to detect the expression levels of the corresponding proteins in the mouse xenograft tissues. These antibodies were the same as those used for western blotting and have been described previously. Tissue samples were incubated in a primary antibody solution overnight at 4 °C. The secondary antibody Biotin Conjugated AffiniPure Donkey Anti-Rabbit IgG (1:200; Wuhan Boster Biological Technology) was incubated for 1 hour . Finally, the tissue slices were visualized with a Nikon ECLIPSE Ti microscope system (Nikon Corporation), and the images were processed using Nikon software (NIS-Elements v.AR4.10, Nikon Corporation). IHC Profiler (an ImageJ plugin) was used to perform automatic quantitative analysis in line with the manufacturer’s instructions.
10
Placental and Embryonic Protein Isolation and Immunoblotting
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Protein was isolated from E13.5 placentas and embryos by acetone
precipitation from RNA-isolation buffer (Buffer RLT or TRK) and resuspending in
SUTEB loading buffer (8M Urea, 1% SDS, 10mM EDTA, 10mM Tris-HCl, pH 6.8).
Protein lysates were run on 4–20% SDS-PAGE acrylamide gels and
transferred to PVDF membrane (Millipore). Immunoblots were probed with anti-Mcm2
(Epitomics/Abcam), anti-Mcm2(Cell Signaling Technology), anti-androgen receptor
(Epitomics/Abcam), anti-SMAD2/3(Cell Signaling), anti-p21(Santa Cruz),
anti-MCM4(Cell Signaling Technology), anti-actin (Sigma). Secondary antibodies
used included goat anti-rabbit-HRP (Cell Signaling) and goat anti-mouse-HRP
(Sigma). Crescendo ECL substrate(Millipore) was used and immunoblots digitally
scanned using a cDigit scanner. Quantification of immunoblots was performed
using ImageStudio software.
precipitation from RNA-isolation buffer (Buffer RLT or TRK) and resuspending in
SUTEB loading buffer (8M Urea, 1% SDS, 10mM EDTA, 10mM Tris-HCl, pH 6.8).
Protein lysates were run on 4–20% SDS-PAGE acrylamide gels and
transferred to PVDF membrane (Millipore). Immunoblots were probed with anti-Mcm2
(Epitomics/Abcam), anti-Mcm2(Cell Signaling Technology), anti-androgen receptor
(Epitomics/Abcam), anti-SMAD2/3(Cell Signaling), anti-p21(Santa Cruz),
anti-MCM4(Cell Signaling Technology), anti-actin (Sigma). Secondary antibodies
used included goat anti-rabbit-HRP (Cell Signaling) and goat anti-mouse-HRP
(Sigma). Crescendo ECL substrate(Millipore) was used and immunoblots digitally
scanned using a cDigit scanner. Quantification of immunoblots was performed
using ImageStudio software.
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