M7001
Manufactured by Agilent Technologies
Sourced in United States
The M7001 is a precision measurement instrument designed for laboratory applications. It provides accurate and reliable measurements of various electrical parameters. The core function of the M7001 is to enable precise data collection and analysis for research and testing purposes.
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Lab products found in correlation
M7240, by Agilent Technologies (4 mentions)
Ncl l egfr 384, by Leica (2 mentions)
A0452, by Agilent Technologies (1 mentions)
S2367, by Agilent Technologies (1 mentions)
M3515, by Agilent Technologies (1 mentions)
Envision reagent, by Agilent Technologies (1 mentions)
Z0622, by Agilent Technologies (1 mentions)
Prolong gold mounting medium, by Thermo Fisher Scientific (1 mentions)
Optiview dab ihc detection kit, by Roche (1 mentions)
Ab16667, by Abcam (1 mentions)
Benchmark xt, by Roche (1 mentions)
Iview dab detection kit, by Roche (1 mentions)
Ap1805a, by Abcepta (1 mentions)
Paraformaldehyde (pfa), by Thermo Fisher Scientific (1 mentions)
Ab69643, by Abcam (1 mentions)
Low melt agar, by Merck Group (1 mentions)
D12384, by Merck Group (1 mentions)
H3136, by Merck Group (1 mentions)
Ab6717, by Abcam (1 mentions)
Ab134154, by Abcam (1 mentions)
16 protocols using m7001
1
Colon Tissue Morphology and Immunohistochemistry
2
IHC for p53 in FFPE Samples
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Immunohistochemistry (IHC) in human samples for p53 immunopositivity, previously associated with TP53 mutation (Pfaff et al., 2010; Tabori et al., 2010 ), was performed on formalin-fixed, paraffin-embedded (FFPE) samples (M7001, Dako) using the Menapath Polymer HRP Detection system (A. Menarini Diagnostics). All samples were analyzed by a neuropathologist, blind to mutation status, and by a nuclear stain algorithm (Spectrum, Aperio Technologies). TP53 mutation status was assessed by direct PCR-based DNA sequence analysis, and one tumor pair was assessed by next-generation sequencing (see Supplemental Experimental Procedures ).
3
Immunohistochemical Profiling of TNBC Subtypes
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Immunohistochemical reactions were performed automatically (Dako Autostainer immunostaining system; Dako) for CK5/6 (D5/16 B4; Dako; 1∶100), EGFR (2-18C9; Dako; 1∶100), AR (F.39.4.1; BioGenex; 1∶100), and p53 (mouse (Dako, M7001; 1∶100). P53 was considered in the analysis as a quality control marker of the immunohistochemical reactions because it is a driver gene in TNBCs [20] (link). Appropriate positive and negative control tissues were run concurrently.
The expression of CK5/6 was cytoplasmic, the expression of EGFR was both cytoplasmic and membranous, expression of AR and p53 was nuclear. Cytoplasmic expression in ≥10% of tumor cells for CK5/6, membranous staining in ≥10% of tumor cells for EGFR, and nuclear staining in ≥5% of tumor cells for AR and ≥50% for p53 were accepted as positive, as previously described [36] (link), [37] (link).
TNBCs were divided into subtypes of breast cancer as defined by their IHC profiles as basal-like triple negative (Core Basal; negative for ER, PR, and HER-2 and positive for CK5/6 and/or EGFR), and five negative (5NP; negative for ER, PR, HER-2, CK5/6, and EGFR). Slides were scored independently by three pathologists (SB, CI, MF) blinded to breast cancer subtype; one pathologist (MF) converted scores to numbers, selected cutoff values for each marker and entered data into Excel files.
The expression of CK5/6 was cytoplasmic, the expression of EGFR was both cytoplasmic and membranous, expression of AR and p53 was nuclear. Cytoplasmic expression in ≥10% of tumor cells for CK5/6, membranous staining in ≥10% of tumor cells for EGFR, and nuclear staining in ≥5% of tumor cells for AR and ≥50% for p53 were accepted as positive, as previously described [36] (link), [37] (link).
TNBCs were divided into subtypes of breast cancer as defined by their IHC profiles as basal-like triple negative (Core Basal; negative for ER, PR, and HER-2 and positive for CK5/6 and/or EGFR), and five negative (5NP; negative for ER, PR, HER-2, CK5/6, and EGFR). Slides were scored independently by three pathologists (SB, CI, MF) blinded to breast cancer subtype; one pathologist (MF) converted scores to numbers, selected cutoff values for each marker and entered data into Excel files.
4
Immunohistochemical Analysis of Cellular Markers
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Immunohistochemistry was performed as previously described [41 (link)]. Tissue sections were blocked with Background Sniper (BS966, Biocare Medical). Antigen Retrieval was performed in Tris/EDTA pH 9 Buffer for 20 minutes (S2367, Dako). The sections were incubated overnight with the appropriate primary antibody: pT356RB1 (1:200, 2223-1, Epitomics), RB1 (1:200, #9309, Cell Signaling Technology), pT202/Y204ERK1/2 (1:100, #9101S, Cell Signaling Technology), N-terminal TP53 (1:200, M7001, DAKO), or Ki-67 (1:800, AC-0009, Epitomics), and pan-cytokeratin (Rabbit 1:400, Z0622, Dako or Mouse, 1:100, M3515, Dako; tumor mask) in Da Vinci Green antibody diluent (PD900, Biocare Medical) at 4°C overnight. Signals were intensified with Envision reagents (DAKO). Pan-cytokeratin primary antibody was probed with an Alexa Fluor 555 dye-labeled secondary antibody (Invitrogen). Primary antibody visualization was accomplished using a Cy-5-tyramide signal amplification system (TSA; AT705A, PerkinElmer). Tissue nuclei were stained using Prolong Gold mounting medium (P36931; Molecular Probes) containing 4,6-diamidino-2-phenylindole (DAPI). HistoRx PM-2000 (HistoRx) with AQUAsition software was used for automated image capture as previously described [41 (link)].
5
Assessing MDM2 and p53 Expression in Tumor Tissue
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We applied immunohistochemical (IHC) staining to formalin-fixed, paraffin-embedded tumor tissue sections (4-μm thick), using the OptiView DAB IHC Detection Kit (Roche) and Ventana automated slide strainers (Roche). The primary antibodies and their dilutions comprised anti-MDM2 diluted to 1:100 (#33–7100, Invitrogen Corporation) and anti-p53 diluted to 1:50 (M 7001, Dako). Stained tissue sections were reviewed and scored by a pathologist (Jen-Chieh Lee) who is an expert in the interpretation of MDM2 expression [28 (link)] and was blinded to the patients’ demographic data and clinical outcomes. Expression was defined as positive when at least 10% of the tumor cells had positive staining [21 (link)]. The positive controls of p53 and MDM2 staining were colon adenoma and liposarcoma, respectively.
6
Immunohistochemical Analysis of CCRCC Biomarkers
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From each case of CCRCCs, two to three 3.0 mm cores were taken from morphologically representative regions of each formalin-fixed paraffin-embedded tumor and precisely arrayed by a manual method. Five-µm-thick sections of the tissue microarray blocks were cut and H&E staining was performed by the usual methods. From each tissue microarray block, 4-µm-thick sections were deparaffinized, rehydrated, and quenched with hydrogen peroxide. Immunohistochemical staining was conducted using Bench Mark XT with iVIEW DAB detection kit (Ventana Medical Systems, Tucson, AZ, USA). The primary antibodies used were IMP3 (1:300, M3626, Dako, Carpinteria, CA, USA), p53 (1:100, M7001, Dako), and Ki-67 (1:200, ab16667, Abcam, San Francisco, CA, USA). The sections were visualized with diaminobenzidine and counterstained with hematoxylin. Tissue of RCC with known IMP expression was used as a positive control. Immunohistochemical staining with primary antibody omitted was used as a negative control.
7
Histological and Immunohistochemical Analysis of Organoids
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Paraffin-embedded biopsies and 3D organoid products were serially sectioned and subjected to hematoxylin and eosin (H&E) staining and immunohistochemistry (IHC) as described previously.13 (link), 46 (link) For IHC, sections were incubated with anti-p53 polyclonal antibody (M7001, 1:200; Dako, Copenhagen, Denmark) or anti-Ki67 monoclonal antibody (M7240, 1:800; Dako), or anticleaved light chain 3 (LC3) polyclonal antibody (AP1805a, 1:250; Abgent, San Diego, CA) overnight at 4°C. H&E stained slides were evaluated by 2 pathologists (SN and AKS) blind to clinical data and conditions. Ki-67 labeling index was determined by counting at least 10 organoids per group.
8
Immunocytochemical Evaluation of Protein Expression
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To evaluate the protein expression, immunocytochemistry (ICC) technique was applied. After 5 days of coculture, the islets in all groups were fixed in 4% paraformaldehyde (Gibco, Germany) and embedded in low melt agar (Sigma, Germany). In the direct cocultured group, both islet and MSCs were fixed and separated from the plate with a scraper. After preparing 5 μm sections and deparaffinization, the slides were subjected to ICC using primary antibodies against Bcl-2 (Abcam, France, #ab115807), Bax (Abcam, France, #ab69643), active caspase-3 (Abcam, France, #ab32042), HIF-1α (Medaysis, USA, #RM0374), and p53 (Dako, USA, #M7001). HRP-secondary antibody (Abcam, France, #ab6717) was used for detection after overnight incubation with primary antibodies. Positive cells for protein expression appeared with 3,3′-diaminobenzidine (Sigma, Germany, #D12384) staining and counterstained by hematoxylin (Sigma, Germany, #H3136). The protein expression rate was calculated by the H-score method using the following formula: H − score = 1 × (%mild staining) + 2 × (%moderate staining) + 3 × (%strong staining) [3 (link), 28 (link)].
9
Immunohistochemical Analysis of TACC3, p53, and Ki-67
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After deparaffinization, antigen retrieval, and endogenous peroxidase blocking, FFPE tissues of 3-μm thicknesses were incubated with TACC3 (rabbit monoclonal, clone: EPR7756; ab134154 [Abcam], 1:200 dilution), p53 (mouse monoclonal, clone: Do-7; M7001 [Dako]), and Ki-67 (mouse monoclonal, clone: MIB-1; M7240, [Dako]) as primary antibodies. Samples were next incubated with Dako REAL EnVision Detection System (Dako) as a secondary antibody. Diaminobenzidine was used to visualize the immunoreaction.
The cytoplasmic staining was defined as TACC3-positive according to the criteria used in other malignancies [10 (link)–18 (link)]. Furthermore, nuclear staining was regarded as positive for p53 and Ki-67 based on previous studies [19 (link), 21 (link), 22 (link)]. The ratios of TACC3-, p53-, and Ki-67-positive tumor cells were calculated by manual counting of at least 100 tumor cells in 5 fields under an optical microscope with 400-fold magnification by the 2 pathologists, who were blinded to any clinical details.
The cytoplasmic staining was defined as TACC3-positive according to the criteria used in other malignancies [10 (link)–18 (link)]. Furthermore, nuclear staining was regarded as positive for p53 and Ki-67 based on previous studies [19 (link), 21 (link), 22 (link)]. The ratios of TACC3-, p53-, and Ki-67-positive tumor cells were calculated by manual counting of at least 100 tumor cells in 5 fields under an optical microscope with 400-fold magnification by the 2 pathologists, who were blinded to any clinical details.
10
Immunohistochemical Profiling of Prostate Tissue
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Tissue sections were cut in 4 µM sections from paraffin blocks and mounted onto slides. Sections underwent preprocessing where they were deparaffinized with xylene and ethanol followed by rehydration and antigen retrieval. Antigen retrieval of the tissue sections was performed using a PT‐Link module (DAKO, Glostrup, Denmark) at 95°C to 99°C for 20 minutes (pH 9.0). The sections were then stained in a DAKO Autostainer‐plus using the EnVision FLEX including Peroxidase‐Blocking Reagent (DAKO). Consecutive sections of the TMAs were immunostained for p63 (M7001, 1:50; DAKO) + AMACR (α‐methyl acyl‐CoA racemase [M3616, 1:100; DAKO], tSTAT3 [8019, 1:50 Santa Cruz Biotechnology, Dallas, TX], pSTAT3 phosphorylated at serine 727 [9134, 1:100; Cell Signaling Technology, Danvers, MA], and pSTAT3 phosphorylated at tyrosine 705 [76315, 1:100; Abcam, Cambridge, UK]). Controls were performed to verify the antibody specificity (Figure S1). The p63/AMACR double staining allowed specific identification of benign versus tumor areas by the visualization of the nuclear p63‐positive basal cells and cytoplasmic AMACR‐positive tumor cells. Examples of the four different immunostainings are shown in Figure 1 .
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