Frozen cross sections of TA muscle were used for succinate dehydrogenase staining. Briefly, slides were incubated in staining buffer containing 0.5 M disodium succinate (Sigma), 20 mM MgCl2, and 0.5 mg/mL of nitro blue tetrazolium (VWR) for 15 min at 37°C. Staining was stopped by immersing the slides in PBS.
Nitro blue tetrazolium
Manufactured by Avantor
Sourced in United States
Nitro blue tetrazolium is a chemical compound used in biochemical assays and laboratory procedures. It serves as a colorimetric indicator, undergoing a color change when reduced, often used to detect the presence of certain enzymes or oxidative processes.
Automatically generated - may contain errors
Lab products found in correlation
5 bromo 4 chloro 3 indolyl phosphate, by Avantor (3 mentions)
Trans blot apparatus, by Bio-Rad (2 mentions)
5 bromo 4 chloro 3 indoyl phosphate, by Avantor (2 mentions)
Quantity one program, by Bio-Rad (2 mentions)
Pgem t plasmid, by Promega (2 mentions)
Digoxigenin rna labeling kit, by Roche (2 mentions)
Disodium succinate, by Merck Group (1 mentions)
Ferrous ammonium sulfate, by Merck Group (1 mentions)
Pyruvate, by Merck Group (1 mentions)
Vit e, by Merck Group (1 mentions)
Sodium hypochlorite, by Merck Group (1 mentions)
3 4 5 dimethylthiazol 2 yl 2 5 diphenyltetrazolium bromide mtt, by Merck Group (1 mentions)
1 1 diphenyl 2 picrylhydrazyl radical dpph, by Merck Group (1 mentions)
Hydrogen peroxide h2o2, by Merck Group (1 mentions)
Alkaline phosphatase conjugated goat anti human igg, by Merck Group (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
5 bromo 4 chloro 3 indolyl phosphate, by Roche (1 mentions)
Kiesel g 60 silica gel resins, by Merck Group (1 mentions)
Deuterated solvent, by Merck Group (1 mentions)
Kieselgel 60 f254, by Merck Group (1 mentions)
10 protocols using nitro blue tetrazolium
1
Succinate Dehydrogenase Staining of TA Muscle
2
Antioxidant and Free Radical Scavenging Assays
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The following reagents were obtained from Sigma-Aldrich (St Louis, MO): ENT, 1,1-Diphenyl-2-picrylhydrazyl radical (DPPH), pyruvate, Vit C, Vit E, 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl-tetrazolium bromide (MTT), iron (III) chloride (FeCl3), EDTA, ferrous ammonium sulfate, hydrogen peroxide (H2O2), sodium hypochlorite and ferrozine. We purchased the following reagents from VWR: 2,2′-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) and nitroblue tetrazolium (NBT). Dihydrorhodamine (DHR123) was obtained from Cayman Chemical (Ann Arbor, MI). Hydroxyphenyl fluorescein (HPF, Hydroxyl/Peroxynitrite Detection Kit™) was obtained from Cell Technology, Inc (Mountain View, CA).
3
Immunoblotting of HCT116 Cells from TME
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Immunoblotting was performed on HCT116 beads from TME cultures as described in detail [41 (link)]. In short, HCT116 were retrieved from alginate beads by dissolving in 55 mM sodium citrate solution for 15 min and subsequent washing with Hank’s solution. Samples were lysed (50 mM Tris/HCl, pH 7.2/150 mM NaCl/(v/v) Triton X-100/1 mM sodium orthovanadate/50 mM sodium pyrophosphate/100 mM sodium fluoride/4 µg/mL pepstatin A/1 mM PMSF) for 30 min on ice to extract whole-cell proteins. Protein content was measured with the bicinchoninic acid system (Uptima, France) using bovine serum albumin (BSA) as standard, proteins reduced with 2-mercaptoethanol and total protein concentrations adjusted (500 ng per lane total protein). After separation of proteins with sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), proteins were blotted onto a nitrocellulose membrane using a transblot apparatus (Bio-Rad, Munich). Membranes were incubated overnight (4 °C) with primary antibodies (1:10.000) and after subsequent washing, incubated for 2 h with alkaline-phosphatase coupled secondary antibodies (1:10.000). Finally, specific binding was detected using nitro blue tetrazolium and 5-bromo-4-chloro-3-indoyl-phosphate (VWR, Darmstadt, Germany) and bands quantified using the Quantity One program (Bio-Rad, Munich). β-Actin was used to normalize samples to control.
4
Whole cell lysate western blot protocol
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Western blot investigations were performed with whole cell lysates as described in [89 (link)]. After dissolving of the alginate, HCT116 cells were lysed (50 mM Tris/HCl, pH 7.2/150 mM NaCl/(v/v) Triton x-100/1 mM sodium orthovanadate/50 mM sodium pyrophosphate/100 mM sodium fluoride/4 µg/mL pepstatin A/1 mM PMSF) on ice for 30 min and total protein content was measured with the bicinchinonic acid system (Uptima, Monlucon, France) using bovine serum albumin as standard. After reduction, proteins were separated by SDS-PAGE electrophoresis, and blotted onto a nitrocellulose membrane (Transblot apparatus, Bio-Rad, Munich, Germany). Membranes were incubated for 2 h with primary antibodies (1:10.000) in blocking buffer (skimmed milk powder 1% in PBS) and for 1.5 h with secondary antibodies (1:10.000) and the specific binding detected using nitro blue tetrazolium and 5-bromo-4-chloro-3-indoyl-phosphate (VWR, Munich, Germany). The bands were quantified by the Quantity One program (Bio-Rad, Munich, Germany) and β-Actin was used to normalize samples to control.
5
Recombinant Protein Immunodetection Assay
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E. coli cells producing the recombinant proteins rED1+2, rED3_301, rED3delA+, and rED3delD+ were harvested by centrifugation and lysed with a buffer (50 mM Tris-HCl, pH 6.8, 200 mM dithiothreitol, and 4% sodium dodecyl sulfate). The lysates were fractioned using 12.5% SDS-PAGE and transferred onto a nitrocellulose membrane (Bio-Rad, Hercules, CA). After blocking with 5% skimmed milk in phosphate-buffered saline (PBS) with 0.05% Tween 20 (PBST), the membrane was incubated with 5 μg/mL chFVN145 at 37°C for 1 h. Immune complexes were detected using alkaline phosphatase-conjugated goat anti-human IgG (Sigma-Aldrich, St. Louis, MO) and visualized by a mixture of nitro blue tetrazolium (Amresco, Radnor, PA) and 5-bromo-4-chloro-3-indolylphosphate (Roche, Basel, Switzerland) for 20 min.
6
Antioxidant Extraction and Analysis
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The distillated organic solvents of n-hexane, ethyl acetic, and methanol were used for sample extraction. For the antioxidant assay, SOD reagents nitro blue tetrazolium (NBT), riboflavin, N,N,N’,N’-tetramethyl ethylenediamine (TEMED), and PBS were purchased from Amresco. The thin-layer chromatography (TLC) spray reagent, 10% sulfuric acid in ethanol, used in this study was purchased from Merck and Sigma-Aldrich. Kiesel G 60 silica gel resins and ODS columns of LiChroprep RP-18 (Merck, Darmstadt, Germany) were used for column chromatography. TLC analysis was performed using Kiesel gel 60 F254 and RP-18 F254S (Merck). Deuterated solvents were purchased from Merck Co., Ltd., and Sigma-Aldrich Co., Ltd. (St. Louis, MO, USA).
7
Identification and Purification of S. thunbergii
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S. thunbergii was purchased from a local store (Dalian, Liaoning Province, China). The material was identified by Professor Liu, Huaqiao University, Fujian, China. Monosaccharide and dextran standard samples (Sigma Co., St Louis, MO, USA), Nitro Blue Tetrazolium (NBT, Amresco Inc., Solon, OH, USA), Nicotinamide Adenine Dinucleotide Hydrogen (NADH, Amresco Inc., Solon, OH, USA), and Phenazine Methosulfate (PMS, Fluka Inc., St Louis, MO, USA) were purchased from a local agent (Taijing Co., Xiamen), Thiazolyl Blue Tetrazolium Bromide (MTT, Sigma Co., St Louis, MO, USA), while DEAE Sepharose CL-6B was acquired from Pharmacia Co. (Sweden). All other reagents used were of analytical grade.
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S. thunbergii was purchased from a local store (Dalian, Liaoning Province, China). The material was identified by Professor Liu, Huaqiao University, Fujian, China. Monosaccharide and dextran standard samples (Sigma Co., St Louis, MO, USA), Nitro Blue Tetrazolium (NBT, Amresco Inc., Solon, OH, USA), Nicotinamide Adenine Dinucleotide Hydrogen (NADH, Amresco Inc., Solon, OH, USA), and Phenazine Methosulfate (PMS, Fluka Inc., St Louis, MO, USA) were purchased from a local agent (Taijing Co., Xiamen), Thiazolyl Blue Tetrazolium Bromide (MTT, Sigma Co., St Louis, MO, USA), while DEAE Sepharose CL-6B was acquired from Pharmacia Co. (Sweden). All other reagents used were of analytical grade.
8
In Situ Hybridization Technique for Detecting Cstb Expression in Mouse Uterus
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In situ hybridization was performed as previously described [38 (link)]. In brief, total RNAs were isolated from the mouse uterus on day 8 of pregnancy and amplified with Cstb primers after reverse transcription. The amplified gene fragment was cloned into the pGEM-T plasmid (Promega) and sequenced. For template preparation, the plasmids were amplified with primers for SP6 and T7. Digoxigenin-labeled antisense or sense complementary RNA probes were transcribed in vitro using a digoxigenin RNA labeling kit (Roche Applied Science, San Francisco, CA, USA), according to the manufacturer’s instructions. Primers used for in situ hybridization were listed in Table 1 . Frozen uterine sections (10 μm) were fixed in 4% paraformaldehyde solution for 1 h. They were hybridized at 55 °C for 16 h. The sections were then incubated in the alkaline phosphatase-conjugated anti-digoxigenin antibody (1:5000; Roche Applied Science, Penzberg, Germany). The positive signal was identified using a buffer containing 0.4 mM 5-bromo-4-chloro-3-indolyl phosphate (Amresco, Solon, OH, USA) and 0.4 mM nitro blue tetrazolium (Amresco, Solon, OH, USA) as a dark brown color. All of the sections were counterstained with 1% methyl green.
9
Paraformaldehyde Fixation and Enzymatic Staining
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Frozen uterine sections were fixed with 4% paraformaldehyde in PBS for 10 min at 4°C and washed with 1 × PBS three times for 5 min. The uterine sections were then washed in PBS, followed by incubation at room temperature in an AP substrate solution containing 5-bromo-4-chloro- 3-indolyl phosphate (Amresco, United Sates) and nitro blue tetrazolium (Amresco, United Sates).
10
In Situ Hybridization of Uterine Tissue
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Each probe’s cDNA template was amplified with the specific primers (listed in Table 2 ) and cloned into a pGEM-T plasmid (Promega, United Sates). Digoxigenin-labeled anti-sense or sense cRNA probes were transcribed in vitro using a digoxigenin RNA labeling kit (Roche Applied Science, Switzerland). In situ hybridization was performed as previously described (Hu et al., 2008 (link)). In brief, uteri were frozen sectioned into 10 mm, and then fixed in 4% (wt/vol) paraformaldehyde, permeabilized, and hybridized with each anti-sense cRNA probe (1:100) at 55°C overnight. A digoxigenin-labeled sense probe was used as the negative control. Following hybridization and post-hybridization washes, sections were incubated in an anti-digoxigenin antibody conjugated with alkaline phosphatase at 4°C overnight (Roche Applied Science, Switzerland). The signal was visualized as dark brown by incubating within a substrate solution containing 5-bromo-4-chloro-3-indolyl phosphate (Amresco, United Sates) and nitro blue tetrazolium (Amresco, United Sates). The activity of endogenous alkaline phosphatase was blocked by levamisole (2 mM, Sigma, United Sates). The sections were counterstained with 1% methyl green.
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