Cells were lysed in a buffer containing 50 mM Tris-HCl (pH 7.5), 200 mM NaCl, 1% Tween-20, 0.2% NP-40, 2 mM PMSF, and 50 mM β-glycerophosphate and protease inhibitor cocktail tablet (Roche). 40μg of protein lysates were loaded into SDS-PAGE mini-gels (Bio-Rad). The gel was then transferred onto nitrocellulose membranes for 2 hours at 100mV. Anti-MLH1 (1:1000) (BD Pharmingen) and anti-β-ACTIN (1:10000) (Sigma Aldrich) antibodies were diluted in Intercept Blocking Buffer (LI-COR Biosciences) and incubated overnight at 4°C. Fluorescent secondary antibody anti-mouse IgG-AlexaFluor488 (LI-COR Biosciences) was diluted (1:10000) in Intercept Blocking Buffer and incubated for 1 hour at room temperature. Image acquisition was perfomed using a Odyssey Clx machine (LI-COR Biosciences).
Anti mlh1
Manufactured by BD
Sourced in United States
Anti-MLH1 is a laboratory reagent used in research applications. It is a monoclonal antibody that specifically binds to the MLH1 protein, which is involved in DNA mismatch repair. The primary function of Anti-MLH1 is to detect and quantify the presence of MLH1 in various biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti msh2, by BD (5 mentions)
Anti pms2, by BD (5 mentions)
Anti msh6, by BD (3 mentions)
Anti β actin, by Merck Group (2 mentions)
Anti sycp1, by Abcam (2 mentions)
Anti msh2, by Merck Group (2 mentions)
Sds page minigels, by Bio-Rad (1 mentions)
Protease inhibitor cocktail tablet, by Roche (1 mentions)
Odyssey clx machine, by LI COR (1 mentions)
Intercept blocking buffer, by LI COR (1 mentions)
Alexa 488, by Thermo Fisher Scientific (1 mentions)
Alexa 488 phalloidin, by Thermo Fisher Scientific (1 mentions)
Prolong glass antifade mountant, by Thermo Fisher Scientific (1 mentions)
Mitotracker red cm h2xros, by Thermo Fisher Scientific (1 mentions)
Alexa 594, by Thermo Fisher Scientific (1 mentions)
Alexa 555, by Thermo Fisher Scientific (1 mentions)
Anti tubulin, by Cell Signaling Technology (1 mentions)
Anti tcfl5, by Merck Group (1 mentions)
Anti sycp3, by Cell Signaling Technology (1 mentions)
Anti pan cadherin, by Cell Signaling Technology (1 mentions)
16 protocols using anti mlh1
1
Western Blot Analysis of MLH1 Protein
2
Molecular Characterization of Right-Sided Colorectal Cancer
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Primary tumor location was determined by operative findings. Cancer in the cecum, ascending colon, hepatic flexure, or transverse colon was classified as right-sided; and cancer in the splenic flexure, descending colon, sigmoid colon, rectosigmoid, or rectum was classified as left-sided [29 (link), 30 (link)]. Histopathological features associated with RCRC, such as medullary type, mucinous type, signet ring type, and tumor-infiltrating lymphocytes were analyzed by a previously reported method [31 ]. MutL homologue 1 (MLH1)/MutS homologue 2 (MSH2) status was evaluated in 110 of the 201 patients by immunohistochemistry with anti-MLH1 (1:50; BD Biosciences PharMingen, San Diego, CA) and anti-MSH2 (1:50; Leica Microbiosystems, Tokyo, Japan) antibodies. Hypermutation was defined as a tumor with MSI-H and/or high tumor mutation burden (TMB), as described previously [25 (link)], using CGS. TMB was calculated as the number of non-synonymous mutations per megabase of sequence in the panel (panel size = 1.3 Mb). To be classified as hypermutated, the threshold of TMB was set as the lowest TMB observed in tumors with MSI-H. Tumors with mutations in POLE or other DNA repair genes can have very high TMB but not show MSI-H [25 (link)].
3
Immunohistochemical Evaluation of MMR Proteins
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Immunohistochemical (IHC) evaluation of MMR proteins expression was carried out on paraffin-embedded tissue sections of all tumors. The following mouse monoclonal antibodies were used: anti-MLH1, anti-MSH2 (PharMingen, San Diego, CA), and anti-MSH6 Transduction Laboratories, BD Biosciences, Brazil. For PMS2 protein, a rabbit monoclonal antibody was used (Ventana Roche Diagnostic, Italy). Immunostaining was executed by the avidin-biotin peroxidase technique; diaminobenzidine was used as a chromogen. Staining was carried out in NEX-ES Automatic Staining System, after counterstaining with Hematoxylin. Normal tissue and stromal cells or lymphocytes adjacent to the respective tumor were used as internal positive controls. Loss of MMR proteins expression was defined as complete absence of nuclear staining in tumor cells (but maintained in normal epithelial and stromal cells).
4
Immunodetection of Meiotic Proteins in Murine Spermatocytes
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Testes from 8 week-mice were decapsulated and seminiferous tubules were fixed and processed for spermatocyte spreading by the drying-down technique previously described20 (link), or processed for squashing as previously reported21 (link). Spread spermatocytes and squashed seminiferous tubules slides were rinsed three times in PBS and blocked with 2% Bovine Serum Albumin (BSA) in PBS for 20 min at room temperature (RT). After blocking, slides were incubated overnight at 4 °C with anti-TCFL5 (Sigma), anti-SYCP3 (Cell Signalling), anti-SYCP1 (Abcam), anti-H1t (Cell Signalling), anti-γH2AX (Cell Signalling), anti-MLH1 (Pharmingen), anti-Pan-cadherin (Cell Signaling) and anti-Tubulin (Cell Signalling) primary antibodies. Then, slides were washed in PBS and incubated for 1 h at RT with their corresponding secondary antibodies against rabbit, mouse, and guinea pig IgGs conjugated with Alexa 555, Alexa 594, and Alexa 488 (Molecular Probes). For actin and flagellar mitochondria immunodetections Alexa 488 Phalloidin (Thermo Fisher) and Mitotracker Red CM-H 2 Xros (Thermo Fisher) were used, respectively. Finally, cells were stained with 1 μg/ml DAPI and mounted using ProLong Glass Antifade Mountant (Thermo Fisher Scientific). Images and stacks were taken in sCMOS camera on a Zeiss Axiovert microscope and analyzed using ImageJ (NIH) and Adobe Photoshop CS5.1 software.
5
Evaluating Mismatch Repair Deficiency
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We used the presence of MSI to assess the loss of function of mismatch repair gene activity. Immunohistochemistry (IHC) and/or Multiplex polymerase chain reaction method was used to evaluate the status of MSI. We conducted IHC analysis of MSH2, MSH6, PMS2, and MLH1 proteins with formalin-fixed paraffin-embedded tumor samples. After the tumor area adjacent to normal mucosa and/or lymphocytic infiltration was marked, 4 mm of paraffinized tissue was removed, and multiple tissue blocks were prepared. Finally, 4-µm thick sections were obtained for IHC following standard protocols. The mouse monoclonal antibodies used were anti-MSH2, anti-MSH6, anti-MLH1, and anti-PMS2 (BD Pharmingen). Tumors showing a proportion of stained nuclei higher than 10% were classified as staining positive; all others were regarded as negative (Figure 1 ).
MSI test was performed by multiplex polymerase chain reaction and analysis with a 3130×1 genetic analyzer. MSI testing of DNA samples was based on five dinucleotide markers (NR27, NR21, BAT26, BAT25, and NR24). Tumors that showed instability in ≥2/5 of markers tested were classified as a high MSI and 1/5 of markers were classified as low MSI. Tumors that showed instability in 0/5 of markers were designated as microsatellite stable (MSS) cancers. Only high MSI cases were considered MSI positive.
MSI test was performed by multiplex polymerase chain reaction and analysis with a 3130×1 genetic analyzer. MSI testing of DNA samples was based on five dinucleotide markers (NR27, NR21, BAT26, BAT25, and NR24). Tumors that showed instability in ≥2/5 of markers tested were classified as a high MSI and 1/5 of markers were classified as low MSI. Tumors that showed instability in 0/5 of markers were designated as microsatellite stable (MSS) cancers. Only high MSI cases were considered MSI positive.
6
Protein Extraction and Western Blotting
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Total protein was extracted using RIPA buffer and was then electrophoresed on SDS-PAGE. Western blotting was conducted with mouse monoclonal anti-E-cadherin (1:500; BD Bioscience), anti-MLH1 (1:500; Pharmingen) and anti-MSH2 (1:500; Merck) antibodies, as has been reported [24 (link)].
7
Quantitative Immunoblot Analysis of MMR Proteins
8
Protein Expression and Immunoblotting
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Cells were lysed in ice-cold lysis buffer containing Hepes 50 nM, pH 7.5, EDTA 1 mM, pH 7, NaCl 150 mM, Naf 100 mM, Na3VO4 1 mM, Triton X-100 1%, and Proteinase Inhibitor Cocktail. Cell lysates were migrated in 12% SDS-PAGE (Sodium Dodecyl Sulfate–PolyAcrylamide Gel Electrophoresis). The following primary antibodies were used: anti-RAD51 (1/1000), anti-BMI1 (#2830, Cell Signaling, 1/1000), anti-LIG4 (MA5-32775, Thermo, 1/1000), anti-ATR (sc1887, Santa Cruz, 1/1000), and anti-phospho-ATR (2853 s, Cell Signaling, 1/1000), anti-ERCC1 (sheep mAb, Gift from C. Lachaud (CRCM, Marseille), 1/1000), and anti-MLH1 (BD Biosciences, 1/1000). Detection of vinculin (V9131, Sigma, 1/2000) or GAPDH (Rabbit pAb, Cell Signaling, 1/5000) was used as loading control.
9
Immunohistochemical Analysis of Mismatch Repair Proteins
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Immunohistochemical (IHC) analysis of MSH2, MSH6, PMS2, and MLH1 proteins was performed as previously described [16 (link), 19 (link), 20 (link)]. Briefly, after the tumor area adjacent to normal mucosa and/or lymphocytic infiltration had been marked, the paraffinized tissue was removed and multiple tissue blocks were prepared. Finally, 4 μm-thick sections were obtained for IHC following standard protocols. The mouse monoclonal antibodies used were anti-MSH2, anti-MSH6, anti-MLH1, and anti-PMS2 (BD Pharmingen, CA, USA).
10
Western Blot Analysis of Protein Expression
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Western blot analysis was performed as described [7 (link)]. Proteins were detected by the Odyssey 9120 Infrared Imaging System (Li-Cor Biosciences, Lincoln, Nebraska, USA). The antibodies used were anti-MGMT (Merck Millipore, Billerica, Massachusetts, USA), anti-HSP90, anti-beta-actin (Santa Cruz Biotechnology, Heidelberg, Germany), anti-MSH2 (Calbiochem, San Diego, CA, USA), anti-MSH6 (Transduction Laboratories, Lexington, KY, USA) and anti-MLH1 (BD Pharmingen, Heidelberg, Germany).
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