Cells were seeded on bone slices and cultured in the inducing medium for 6 days. Then bone slices were incubated in 0.5 N NaOH for 1 min and cells were scraped off using a teeth brush. After fully washing with PBS, the bone slices were incubated with 20 mg/mL peroxidase-conjugated wheat germ agglutinin in PBS (Sigma) for 1 h, and exposed to SIGMAFASTTM 3,3'-Diaminobenzidine tablets (Sigma) for 10 min.
Sigmafast 3 3 diaminobenzidine tablets
Manufactured by Merck Group
Sourced in United States
SIGMAFAST 3,3′-Diaminobenzidine tablets are a laboratory product manufactured by Merck Group. The tablets are used as a chromogenic substrate in immunohistochemistry and enzyme-linked immunosorbent assay (ELISA) applications.
Automatically generated - may contain errors
Lab products found in correlation
Vectastain elite abc kit, by Vector Laboratories (3 mentions)
Goat anti rabbit horseradish peroxidase linked antibodies, by Promega (3 mentions)
Vectastain elite abc hrp kit, by Vector Laboratories (2 mentions)
Peroxidase conjugated wheat germ agglutinin, by Merck Group (1 mentions)
Ridascreen fast gliadin, by R-Biopharm (1 mentions)
Immobilon psq, by Merck Group (1 mentions)
Infinite 200 pro, by Tecan (1 mentions)
Christ alpha 1 2 ld plus, by Martin Christ (1 mentions)
Avidin biotin peroxidase complexes, by Vector Laboratories (1 mentions)
Trupage precast gels, by Merck Group (1 mentions)
Goat anti mouse hrp, by Merck Group (1 mentions)
Clone de u 10, by Merck Group (1 mentions)
Seeblue pre stained standard, by Thermo Fisher Scientific (1 mentions)
Azocasein, by Merck Group (1 mentions)
Seeblue plus2 pre stained standard, by Thermo Fisher Scientific (1 mentions)
O phenylenediamine, by Merck Group (1 mentions)
Invitrolon pvdf filter paper sandwiches, by Thermo Fisher Scientific (1 mentions)
Peptide calibration standard 2, by Bruker (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Hydrogen peroxide, by Merck Group (1 mentions)
28 protocols using sigmafast 3 3 diaminobenzidine tablets
1
Bone Resorption Assay with WGA Staining
2
Protein Characterization Protocol
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Molecular weight protein markers (FERMENTAS Int. Inc., Canada), the polyclonal Anti-gluten wheat rabbit antibody (USBiological, USA), the Anti-rabbit HRP antibody (BD Pharmingen, USA), SIGMAFASTTM 3,3'-Diaminobenzidine tablets (SIGMA, USA), RI-DASCREEN® Fast Gliadin (R-BIOPHARM, Germany), the PVDF transfer membrane ImmobilonPSQ (Millipore, USA), the amino acid standard solution for calibration of amino acid analyzers (Sigma, USA). All other analytical grade chemicals were from Sigma (USA), Fluka (Switzerland) or Applichem (Germany).
Lyophylizer Christ®Alpha1-2 LD Plus (Martin Christ, Germany), the electrophoresis and transfer equipment (BIO-RAD Laboratories Inc., USA), the amino acid analyzer AAA400 (Ingos, Czech Republic), the ELISA reader TECAN Infi nite 200 PRO (TECAN, Switzerland) .
Lyophylizer Christ®Alpha1-2 LD Plus (Martin Christ, Germany), the electrophoresis and transfer equipment (BIO-RAD Laboratories Inc., USA), the amino acid analyzer AAA400 (Ingos, Czech Republic), the ELISA reader TECAN Infi nite 200 PRO (TECAN, Switzerland) .
3
Immunohistochemical Staining of Key Markers
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Rabbit antibodies (Abs) against human Ki67 (Ki67, Thermo Fisher Scientific, Inc, Waltham, MA), human CD44 (CD44, Acris Antibodies, Inc, Rockville, MD), mouse CD31 (CD31, Abcam, Inc, Cambridge, MA), or alpha smooth muscle actin (specific to human and mouse, α-SMA, Acris Antibodies, Inc, Rockville, MD), and biotinylated anti-rabbit Ig secondary Abs (Vector Laboratories, Burlingame, CA) were used for IHC staining. Reagents used were avidin-biotin-peroxidase complexes (Vector Laboratories) and Sigma FAST 3,3-diaminobenzidine tablets (Sigma-Aldrich).
4
Western Blot Protein Detection Protocol
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Cell lysates were mixed with SDS-PAGE sample buffer (with or without β-mercaptoethanol to create reducing and non-reducing conditions, respectively), denatured by boiling for 5 min, and separated on 4–20% TruPAGE pre-cast gels (Sigma-Aldrich). Proteins were transferred onto a nitrocellulose transfer membrane (NitroBind, MSI, Cole-Parmer, IL, United States) using the Bio-Rad (Hercules, CA, United States) wet transfer blotting module. The membrane was subsequently blocked in 5% skim milk (Coles Supermarkets, Canberra, Australia) in tris-buffered saline with 0.1% Tween-20 (TBST) for 2 h at room temperature. Incubation with primary antibodies was performed overnight at 4°C in TBST. Incubation with secondary antibodies conjugated to horseradish peroxidase was performed for 1 h at room temperature in TBST. SIGMAFAST 3,3′-diaminobenzidine tablets (Sigma-Aldrich) were used for visualization of bands according to the manufacturer’s instructions. Staining of β-actin was used as a loading control where appropriate.
5
Myofibril Protein Isolation and Analysis
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Breast myofibrils were prepared by the method of Huff-Lonergan et al. (1995 (link)). The myofibril protein concentration was determined by the method of Robson et al. (1968 (link)). Myofibril samples for SDS-PAGE were prepared by the method of Fritz and Greaser (1991 ). The SDS-PAGE procedures for myofibrils on 12% slab gels (acrylamide: methylenebisacrylamide = 37.5: 1, w/w) was done via the method of Laemmli (1970 (link)). The same amount of protein (250 µ g) from each sample including a pooled 0-day CG sample, which was used as the standard reference, was loaded onto each gel. A pre-stained marker that contained 9 polypeptides in the range of 4 to 250 kDa (SeeBlue Pre-stained Standard, LC5625, Invitrogen Co., Carlsbad, CA) was used as a protein standard. SE 400 slab gel electrophoresis units (Hoefer Scientific Instruments, San Francisco, CA) were used to run all gels with 15 mA at 25°C. After electrophoresis, proteins were transferred from the gel to a nitrocellulose membrane. Western blot analysis was done by the method of Liao and Chou (2014 (link)). A monoclonal primary antibody to desmin (Clone DE-U-10, 1:500 dilution, Sigma-Aldrich Company, St. Louis, MO) and a secondary antibody (goat anti-mouse-HRP, 1:1,000 dilution, Sigma-Aldrich Company) was used to label the blots. Color was developed by using SIGMAFAST 3,3′-diaminobenzidine tablets (Sigma-Aldrich Company).
6
Enzymatic Assay for Protease Activity
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Azocasein, horseradish peroxidase, hyaluronan, hydrogen peroxide, l -leucine, Nα-p-tosyl-l -arginine methyl ester, o-phenylenediamine, peroxidase-labelled anti-horse IgG antibody, phosphate buffered saline with Tween, and Sigma FAST™ 3,3’-diaminobenzidine tablets were purchased from Sigma-Aldrich (St. Louis, MO, USA). Coomassie Brilliant Blue G-250 and trypsin were obtained from Thermo Scientific Pierce (Waltham, MA, USA). NuPAGE MES gel and SeeBlue Plus2 Pre-Stained standard were acquired from Invitrogen (Darmstadt, Germany).
The following chemicals and materials were purchased from different suppliers: anti-bothropic serum (Fundação Ezequiel Dias, Belo Horizonte, MG, Brazil), cisplatin (Incel®, Darrow, Rio de Janeiro, RJ, Brazil), Peptide Calibration Standard II™ (Bruker Daltonics, Bremen, Germany), PVDF membrane (Invitrolon™ PVDF/Filter Paper Sandwiches; Invitrogen™, Carlsbad, CA, USA), trichloroacetic acid (Thermo Fisher Scientific, Waltham, MA, USA), and ZipTip® pipette tips with C18 reversed-phase resin (Millipore, Darmstadt, Germany).
The following chemicals and materials were purchased from different suppliers: anti-bothropic serum (Fundação Ezequiel Dias, Belo Horizonte, MG, Brazil), cisplatin (Incel®, Darrow, Rio de Janeiro, RJ, Brazil), Peptide Calibration Standard II™ (Bruker Daltonics, Bremen, Germany), PVDF membrane (Invitrolon™ PVDF/Filter Paper Sandwiches; Invitrogen™, Carlsbad, CA, USA), trichloroacetic acid (Thermo Fisher Scientific, Waltham, MA, USA), and ZipTip® pipette tips with C18 reversed-phase resin (Millipore, Darmstadt, Germany).
7
Immunohistochemical Staining Protocol
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On the following day, slides were washed with PBS and incubated with secondary biotinylated antibody diluted in 0.5% triton-PBS and 2% for 1.5 h in a humid chamber in RT. The slides were then washed and incubated with AB Solution (VECTASTAIN Elite ABC Hrp Kit, Vector Laboratories, PK6100) for 1 h. Next, slides were washed again in PBS and incubated with DAB substrate (SIGMAFAST™ 3,3′-Diaminobenzidine tablets, Sigma D4293). Finally, the slides were washed again in PBS and covered with DPX mountant for histology (Sigma 4451).
8
Evaluating Blood-Brain Barrier Permeability
9
GFAP Immunohistochemistry in Tissue Sections
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For GFAP immunohistochemistry, tissue was cryosectioned to 40 μm and floating sections were incubated overnight at 4°C in an antibody to GFAP (mouse monoclonal anti-GFAP, Sigma-Aldrich, G3893) in a 1:1000 dilution under conditions including permeabilization with 0.3% Triton X-100 (Sigma-Aldrich, X-100), blocking with 1% bovine serum albumin (Sigma-Aldrich, A9647) and processing with the Vectastain Elite ABC HRP kit (Vector Laboratories, PK-6100, Burlingame, CA, USA). Tissue was then incubated for 1 h at room temperature in biotinylated horse anti-mouse IgG antibody, rat adsorbed, used in a 1:500 dilution (Vector Laboratories, BA-2001), then stained with SIGMAFAST™ 3,3′-diaminobenzidine tablets (Sigma-Aldrich, D4418).
10
Quantitative Analysis of Lung Metastases
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Mammary tumors and lungs were collected and processed as previously described [32] (link), [33] . Immunohistochemistry was performed as previously described [32] (link). The number of lung metastases was determined in two independent histological sections from each mouse and then averaged. Primary antibodies were used at a dilution of 1∶200 and were obtained from the following sources, anti-Ki67, anti-cytokeratin 5 and anti-cytokeratin 14 (Abcam, Cambridge, MA), anti-cytokeratin 18 (Research Diagnostics Inc, Flanders, NJ), and anti-cytokeratin 8 (Fitzgerald Industries International Inc, Concord, MA). Primary antibodies were detected using a 1∶200 dilution of the appropriate secondary antibody and Sigma Fast 3,3′-diaminobenzidine tablets (Sigma, St. Louis, MO). Ki67 immunohistochemistry was quantified using Positive Pixel Count software v9 (Aperio, Vista, CA) following slide scanning on a ScanScope CS slide scanner (Aperio, Vista, CA).
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