As part of the OncoPlus panel, a microsatellite instability (MSI) detection module using data from 336 incidentally captured homopolymers across the 1,213 captured genes was utilized as previously described12 (link). Due to the impact of pre-analytic factors on the MSI-calling module and the inclusion of many FFPE blocks in this study that were > 5 years old, we required that > 90% of microsatellite loci reach the minimum sequencing depth threshold (50X coverage) for quality control and accurate MSI calling. Tumors with < 9% unstable loci were classified as microsatellite-stable, 9 to 15% as indeterminate, and > 15% as unstable. Immunohistochemistry for MSH6 and PMS2 was performed on all tumors classified as indeterminate or unstable, as well as in those without available microsatellite information. PEComas were considered mismatch repair protein-deficient if there was absence of nuclear staining in tumor cells for either MSH6 (rabbit monoclonal EPR3945, dilution 1:200; Abcam, Cambridge, MA) or PMS2 (rabbit monoclonal EPR3945, dilution 1:50; Abcam, Cambridge, MA). For all other staining patterns, the tumor was considered mismatch repair protein-proficient.
Epr3945
Manufactured by Abcam
Sourced in United Kingdom
EPR3945 is a recombinant anti-Coronavirus SARS-CoV-2 nucleocapsid protein antibody. It is produced in E. coli and purified by protein A affinity chromatography.
Automatically generated - may contain errors
Lab products found in correlation
6 protocols using epr3945
1
Microsatellite Instability Detection Protocol
2
Microsatellite Instability Detection Protocol
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As part of the OncoPlus panel, a microsatellite instability (MSI) detection module using data from 336 incidentally captured homopolymers across the 1,213 captured genes was utilized as previously described12 (link). Due to the impact of pre-analytic factors on the MSI-calling module and the inclusion of many FFPE blocks in this study that were > 5 years old, we required that > 90% of microsatellite loci reach the minimum sequencing depth threshold (50X coverage) for quality control and accurate MSI calling. Tumors with < 9% unstable loci were classified as microsatellite-stable, 9 to 15% as indeterminate, and > 15% as unstable. Immunohistochemistry for MSH6 and PMS2 was performed on all tumors classified as indeterminate or unstable, as well as in those without available microsatellite information. PEComas were considered mismatch repair protein-deficient if there was absence of nuclear staining in tumor cells for either MSH6 (rabbit monoclonal EPR3945, dilution 1:200; Abcam, Cambridge, MA) or PMS2 (rabbit monoclonal EPR3945, dilution 1:50; Abcam, Cambridge, MA). For all other staining patterns, the tumor was considered mismatch repair protein-proficient.
3
Western Blot Analysis of DNA Mismatch Repair Proteins
4
Western Blot Analysis of DNA Mismatch Repair Proteins
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Equal amounts of protein were loaded into 4–20% acrylamide gels
(Bio-Rad) and transferred to nitrocellulose membrane. PBS with 5% (w/v) nonfat
dried milk and 0.1% Tween-20 was used to block for 2 h at 4°C. Primary
antibodies were then incubated with the membrane overnight at 4°C.
Antibodies used were rabbit anti-MSH2 (Cell-Signaling Technology D24B5), rabbit
anti-MSH6 (Abcam EPR3945), mouse anti-HA (PY102, gift from Thomas Moran at Mt.
Sinai), rabbit anti-Mx1 (Abcam 95926), and mouse anti-α-Tubulin (Sigma
T5168). Membranes were washed five times in PBS with 0.1% Tween-20 and then
anti-mouse-HRP or anti-rabbit-HRP secondary antibody was added for 1 h. The
membrane was then washed five times and Clarity or Clarity Max ECL substrate
(Bio-Rad) was added before being exposed to film and developed. Uncropped scans
of all Western blots are displayed inSupplementary Figure 6 .
(Bio-Rad) and transferred to nitrocellulose membrane. PBS with 5% (w/v) nonfat
dried milk and 0.1% Tween-20 was used to block for 2 h at 4°C. Primary
antibodies were then incubated with the membrane overnight at 4°C.
Antibodies used were rabbit anti-MSH2 (Cell-Signaling Technology D24B5), rabbit
anti-MSH6 (Abcam EPR3945), mouse anti-HA (PY102, gift from Thomas Moran at Mt.
Sinai), rabbit anti-Mx1 (Abcam 95926), and mouse anti-α-Tubulin (Sigma
T5168). Membranes were washed five times in PBS with 0.1% Tween-20 and then
anti-mouse-HRP or anti-rabbit-HRP secondary antibody was added for 1 h. The
membrane was then washed five times and Clarity or Clarity Max ECL substrate
(Bio-Rad) was added before being exposed to film and developed. Uncropped scans
of all Western blots are displayed in
5
Immunostaining of FFPE Tumor Specimens
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Formalin-fixed paraffin-embedded (FFPE) tumor specimens were prepared by the standard procedures in Department of Diagnostic Pathology, Kyoto University Hospital. A board-certificated pathologist selected multiple and separate cancer lesions from a single tumor. Specimens of FFPE cancer spheroids were prepared as previously reported [14 (link)]. These specimens were sectioned at 4-μm thickness, and stained with H&E or immunostained for MLH-1 (M1, Ventana, Tucson, AZ, USA), MSH2 (G219-1129, Ventana), MSH6 (EPR3945, Abcam, Cambridge, UK), or PMS2 (EPR 3947, Ventana) followed by hematoxylin counterstaining for nuclei.
6
Protein Analysis by Western Blotting
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For protein analysis, the normalized lysates were separated on NuPAGE™ gels (4–12%) under reducing conditions using the MES SDS Running Buffer system. Subsequently, the proteins were transferred to PVDF membranes using the iBlot™ 2 Gel Transfer system. To block non-specific binding, the membranes were incubated in 5% milk in TBS-T (TBS with 0.05% Tween) and then probed with primary antibodies, which were diluted in 5% milk in TBS-T. The primary antibodies used included 1F6 from Millipore Sigma for MSH3, EPR21017-2 from Abcam for MSH2, EPR3945 from Abcam for MSH6, and C4 from Millipore Sigma and 13E5 from Cell Signaling for actin. Bound antibodies were detected using enhanced chemiluminescence (ECL Prime, Cytiva) with a ChemiDoc MP imaging system (BioRad) and quantified by densitometry using Image Lab software 6.1 (BioRad). Each sample was quantified from at least two independent gels (unless indicated otherwise) with different loading orders to minimize bias. Furthermore, all samples from the control group were loaded on each gel for normalization purposes (unless indicated otherwise).
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