anti mouse mgmt monoclonal antibody  (Novus Biologicals)

Journal: Surgical Neurology International

Article Title: Analysis of a synchronous gliosarcoma and meningioma with long survival: A case report and review of the literature

doi: 10.4103/2152-7806.122229

Figure Lengend Snippet: Methylation-specific PCR (MSP) analyses of the MGMT promoter of the three lesions; P (primary gliosarcoma); R (recurrent gliosarcoma); M (meningioma). MSP controls reactions consisted of blood-extracted DNA from a cancer-free individual to use as umethylated DNA control (Un.), and a CpGenome™ Universal Methylated DNA (Chemicon International) as methylated DNA control (m). PCR reactions in the absence of DNA (water) were performed as negative controls for both the unmethylated and methylated reactions

Article Snippet: We further investigated for the presence of MGMT protein expression by immunohistochemistry, using the mouse anti-MGMT monoclonal antibody (dilution 1:400; clone MT3.1, Chemicon International) and correlated with MGMT gene promoter hypermethylation, assessed by methylation-specific PCR (MSP) as previously described.

Techniques: Methylation

Journal: Surgical Neurology International

Article Title: Analysis of a synchronous gliosarcoma and meningioma with long survival: A case report and review of the literature

doi: 10.4103/2152-7806.122229

Figure Lengend Snippet: Imunohistochemistry and Chromogenic In Situ Hybridization (CISH) analysis of the three lesions. COX2 immunohistochemistry (×200) positive for the meningioma lesion and negative for the primary and recurrent gliosarcoma. MGMT staining (×200) was only positive for the meningioma. EGFR immunostaining was negative in primary gliosarcoma and meningioma, with recurrent gliosarcoma exhibiting strong positivity. CISH analysis of EGFR confirmed these findings after EGFR amplification

Article Snippet: We further investigated for the presence of MGMT protein expression by immunohistochemistry, using the mouse anti-MGMT monoclonal antibody (dilution 1:400; clone MT3.1, Chemicon International) and correlated with MGMT gene promoter hypermethylation, assessed by methylation-specific PCR (MSP) as previously described.

Techniques: Chromogenic In Situ Hybridization, Immunohistochemistry, Staining, Immunostaining, Amplification

Journal: Frontiers in Oncology

Article Title: Cyclooxygenase-2 Upregulated by Temozolomide in Glioblastoma Cells Is Shuttled In Extracellular Vesicles Modifying Recipient Cell Phenotype

doi: 10.3389/fonc.2022.933746

Figure Lengend Snippet: Influence of TMZ on T98G TMZ-resistant cells. Cells were daily incubated with increasing doses of TMZ (5-20 μM) for 5 days and (A) cell number was detected by Trypan blue staining. All values are given as fold increase vs. initial time (T0). The results, derived from three experiments performed in duplicate, are expressed as mean ± SEM. The one-way analysis of variance (ANOVA) followed by Dunnet post hoc test show not significant differences. Influence of TMZ on (B) COX-2, (C) β-catenin and (D) MGMT levels was assessed by Western blotting in the presence of vehicle (CNTR), or TMZ, as previously described. The obtained values were normalized vs. β-actin and presented as fold increase vs. CNTR. Data are from three independent experiments and values are expressed as mean ± SEM. For comparative analysis of data, a one-way ANOVA with Dunnet post hoc test was used (*P<0.05; **P<0.01; *** P<0.001 vs. CNTR). Representative images of each immunoblotting are shown. (E) Influence of the COXIB combined with TMZ on COX-2 levels was verified by Western blotting assay in T98G cells daily incubated or not (CNTR) with Celecoxib (CXB) (8 μM), NS398 (20 μM), TMZ (10 μM) or with the co-treatments (CXB+TMZ and NS398+TMZ) for 5 days. Densitometric analysis was performed by normalizing vs. β-actin and presented as fold increase vs. CNTR. Data from three independent experiments are expressed as mean ± SEM. Representative images of each immunoblotting are shown. C+ = positive control (not treated T98G cells). (F) PGE2 levels (fold increase vs. CNTR) released by T98G treated as above described, were assayed by ELISA kit. Results are expressed as mean ± SEM of three experiments in duplicates. (G) Effect of COXIB, CXB and NS398, TMZ and drug combinations on T98G cell number was evaluated by Trypan blue staining. All values are given as fold increase vs. CNTR of two independent experiments performed in triplicate (mean ± SEM). For comparative analysis of data groups, a one-way ANOVA with Tukey post hoc test was used (*P<0.05; **P<0.01; ***P<0.001; ****P<0.0001).

Article Snippet: To remove non-specific binding sites, membranes were incubated with 5% non-fat dry milk in Tris buffered saline for 1 h at room temperature and then incubated overnight at 4°C with primary antibodies: rabbit monoclonal anti-COX-2 (Cell Signaling Technology, Danvers, MA, USA; dilution 1:1000), mouse monoclonal antibody anti-MGMT (BD Biosciences, San José, CA, USA; dilution 1:500), rabbit polyclonal anti-β-catenin antibody (Cell Signaling Technology, Danvers, MA, USA; dilution 1:1000), and mouse monoclonal antibody for anti-β-actin (Bio-Rad, Hercules, CA, USA; dilution 1:1000).

Techniques: Incubation, Staining, Derivative Assay, Western Blot, Positive Control, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Oncology

Article Title: Cyclooxygenase-2 Upregulated by Temozolomide in Glioblastoma Cells Is Shuttled In Extracellular Vesicles Modifying Recipient Cell Phenotype

doi: 10.3389/fonc.2022.933746

Figure Lengend Snippet: Influence of TMZ on U87MG TMZ-sensitive cells. Cells were daily incubated with increasing doses of TMZ (5-20 μM) for 5 days and (A) cell number was detected by Trypan blue staining. All values are given as fold increase vs. initial time (T0). The results, derived from three experiments performed in duplicate, are expressed as mean ± SEM. The one-way analysis of variance (ANOVA) followed by Dunnet post hoc test show not significant differences. Effect of TMZ on (B) COX-2, (C) β-catenin and (D) MGMT levels was assessed by Western blotting in the presence of vehicle (CNTR), or TMZ, as previously described. The obtained values were normalized vs. β-actin and presented as fold increase vs. CNTR. Data are from three independent experiments and values are expressed as mean ± SEM. For comparative analysis of data, a one-way ANOVA with Dunnet post hoc test was used (*P<0.05; **P<0.01; *** P<0.001 vs. CNTR). Representative images of each immunoblotting are shown. (E) Influence of the COXIB combined with TMZ on COX-2 levels was verified by Western blotting assay in U87MG cells daily incubated or not (CNTR) with Celecoxib (CXB) (8 μM), NS398 (20 μM), TMZ (10 μM) or with the co-treatments (CXB+TMZ and NS398+TMZ) for 5 days. Densitometric analysis was performed by normalizing vs. β-actin and presented as fold increase vs. CNTR. Data from three independent experiments are expressed as mean ± SEM. Representative images of each immunoblotting are shown. C+ = positive control (not treated T98G). (F) PGE2 levels (fold increase vs. CNTR) released by U87MG treated as above described, were assayed by ELISA kit. Results are expressed as mean ± SEM of three experiments in duplicates. (G) Effect of COXIB, CXB and NS398, TMZ and drug combinations on U87MG cell number was evaluated by Trypan blue staining. All values are given as fold increase vs. CNTR of two independent experiments performed in triplicate (mean ± SEM). For comparative analysis of data groups, a one-way ANOVA with Tukey post hoc test was used (*P<0.05; **P<0.01; ***P<0.001; ****P<0.0001).

Article Snippet: To remove non-specific binding sites, membranes were incubated with 5% non-fat dry milk in Tris buffered saline for 1 h at room temperature and then incubated overnight at 4°C with primary antibodies: rabbit monoclonal anti-COX-2 (Cell Signaling Technology, Danvers, MA, USA; dilution 1:1000), mouse monoclonal antibody anti-MGMT (BD Biosciences, San José, CA, USA; dilution 1:500), rabbit polyclonal anti-β-catenin antibody (Cell Signaling Technology, Danvers, MA, USA; dilution 1:1000), and mouse monoclonal antibody for anti-β-actin (Bio-Rad, Hercules, CA, USA; dilution 1:1000).

Techniques: Incubation, Staining, Derivative Assay, Western Blot, Positive Control, Enzyme-linked Immunosorbent Assay