Animal experiments were performed as previously described [22 (link)]. Expressions of SDC2 protein were measured using immunochemical analysis on 5-mm slices of formalin fixed paraffin-embedded tumor xenografts in nude mice. Antigens were retrieved by pretreating dewaxed sections in a microwave oven at 750 W for 5 min in a citrate buffer (pH 6) processed with the Super Sensitive Link-Labeled Detection System (Biogenex, Menarini, Florence, Italy). The enzymatic activities were developed using 3-amino-9-ethylcarbazole (Dako, Milan, Italy) as a chromogenic substrate. Following counterstaining with Mayer hematoxylin (Invitrogen), slides were mounted in aqueous mounting medium (glycergel, Dako). Pictures were taken using a LEICA DM 4000B microscope, while the relative level of each protein was calculated using LEICA software, percentage of the mock over the chemotherapeutic treated tumors was calculated and plotted.
Mayer hematoxylin
Manufactured by Thermo Fisher Scientific
Sourced in United States
Mayer's hematoxylin is a staining solution used in histology and cytology to stain cell nuclei. It is a commonly used nuclear stain that binds to the DNA and RNA in cell nuclei, resulting in a blue-purple color.
Automatically generated - may contain errors
Lab products found in correlation
Glycergel, by Agilent Technologies (2 mentions)
3 amino 9 ethylcarbazole, by Agilent Technologies (2 mentions)
Super sensitive link labeled detection system, by BioGenex (2 mentions)
Avidin dh biotinylated horseradish peroxidase h complex with 3 3 diaminobenzidine, by Polysciences (2 mentions)
Dm4000b, by Leica (1 mentions)
Coomassie brilliant blue r 250, by Bio-Rad (1 mentions)
Clusterin, by R&D Systems (1 mentions)
Gelsolin, by Abcam (1 mentions)
Trypsin, by Bio-Rad (1 mentions)
Casein, by Bio-Rad (1 mentions)
Vectastain universal kit hrp, by Vector Laboratories (1 mentions)
Bx51 microscope, by Olympus (1 mentions)
Vectastain elite abc reagent, by Vector Laboratories (1 mentions)
F4 80, by Bio-Rad (1 mentions)
Arginase 1, by Santa Cruz Biotechnology (1 mentions)
P stat3, by Abcam (1 mentions)
6 protocols using mayer hematoxylin
1
Measuring SDC2 Protein in Tumor Xenografts
2
Immunochemical Analysis of DLL1 Protein
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The expression of DLL1 protein was measured using immunochemical analysis of 5-mm slices of formalin-fixed paraffin-embedded tumor xenografts from the nude mice. The tissue slides from all six groups were placed on a single slide and simultaneously subjected to the same immunestaining. Antigens were retrieved by pretreating dewaxed sections in a microwave oven at 750 watts for 5 min in citrate buffer (pH 6) and processed with the Super Sensitive Link-Labeled Detection System (Biogenex, Menarini, Florence, Italy). The enzymatic activities were determined using 3-amino-9-ethylcarbazole (Dako, Milan, Italy) as a chromogenic substrate. Following counterstaining with Mayer hematoxylin (Invitrogen), the slides were mounted in aqueous mounting medium (glycergel, Dako). Pictures were taken usinga LEICA DM 4000B microscope. Accordingto the rates of positive cell in each field, we marked the chipsas 0 (pigment free), + (light yellow),++(yellow),+++ (51∼75%), and ++++(brownish yellow) by their color intensity52 (link).
3
Proteomic Analysis of Cell Lines
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The following reagents were purchased from Sigma-Aldrich Co. (St Louis, MO, USA): Coomassie Brilliant Blue R-250, dithiothreitol (DTT), urea, trypsin, glycerol, glacial acetic acid, alpha-cyano-4-hydroxycinnamic acid, acetonitrile, sodium carbonate, HAuCl4, casein, and Ponceau S. We have purchased ReadyStrip (IPG strip pH 4–7) from Bio-Rad Laboratories Inc. (Hercules, CA, USA). Primary mouse monoclonal antibody or rabbit polyclonal antibodies against gelsolin, calpain, peroxire-doxin-2 (Abcam, Cambridge, UK), PEBP, LDH-B, crystalline (Abgent, San Diego, CA, USA), anexxin-2 and LXRα and CRY7B1 (Novus, Littleton, CA, USA), and clusterin (R&D systems, Inc., Minneapolis, MN, USA) were used. We have purchased Mayer hematoxylin from Richard-Allan Scientific (Thermo Fisher Scientific, Waltham, MA, USA) and immunohistochemistry (IHC) reagents from Biocare Medical (Concord, CA, USA). All cell lines were purchased from the American Type Culture Collection (ATCC) (Manassas, VA, USA).
4
Immunohistochemical Analysis of Tumor Markers
5
Immunohistochemical Analysis of Mouse Tumor Samples
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Tumors and lung samples were dissected from mice, fixed in formalin, and embedded in paraffin. Standard IHC techniques were used according to the manufacturer’s recommendations (Vector Laboratories) by using the primary antibodies against p-STAT3 (Abcam, 1:200), Ki67 (Neomarkers, 1:100), CD31 (Santa Cruz, 1:100), F4/80 (AbD Serotec, 1:50), and arginase1 (Santa Cruz (1:200) for overnight at 4°C. Vectastain Elite ABC reagents (Vector Laboratories) with avidin DH:biotinylated horseradish peroxidase H complex with 3,3′-diaminobenzidine (Polysciences) and Mayer hematoxylin (Fisher Scientific) were used for detection of the bound antibodies. The stained cells were counted in four different fields by using a bright-field microscope in each experimental group, and the average was calculated.
6
Evaluating TRPV2 Expression in TNBC
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Antibody against TRPV2 (1:100, Sigma Aldrich) was applied to formalin fixed paraffin embedded tissues. Four micron sections were incubated with the primary antibody overnight at 4°C. Vectastain Elite ABC reagents with avidin DH:biotinylated horseradish peroxidase H complex with 3,3′-diaminobenzidine (Polysciences) and Mayer hematoxylin (Fisher Scientific) have been used for detection of the bound primary antibodies. Tumors samples were fixed, and embedded in paraffin. We performed tissue microarrays (TMA) staining to stain normal (n=10) and TNBC patient breast tissues (n=116 patients who were treated with multi-agent chemotherapy. TNBC patients’ recurrence free survival (RFS) has been documented at OSU medical center and analyzed after staining with Anti-TRPV2 antibody).
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