NRK-52E cells that had undergone various treatments were fixed in 4% paraformaldehyde and per-mobilized in 0.1% Triton X-100, before they were, respectively, treated with primary mouse monoclonal anti-E-cadherin (E-ca) antibody (Abcam ab1416, 1:100), mouse monoclonal collagen I antibody (Abcam ab260043, 1:100), TLR4 (Abcam ab22048, 1:200), NF-κB (Abcam ab194726, 1:200). After three washes with PBS, the sections were incubated for 2 h with DAR-FITC (1:50) and Texas Red-DAM (1:50) at RT. The fluorescent images were visualized with a Fluoview 300 fluorescence microscope (Olympus, Tokyo, Japan).
Ab194726
Manufactured by Abcam
Sourced in United States, United Kingdom, China
Ab194726 is a laboratory equipment product offered by Abcam. It is a multipurpose device designed for scientific research applications. The core function of this product is to facilitate specific experimental tasks, but a detailed description cannot be provided while maintaining an unbiased and purely factual approach.
Automatically generated - may contain errors
Lab products found in correlation
Ab16502, by Abcam (5 mentions)
Ab205718, by Abcam (5 mentions)
Ab9324, by Abcam (2 mentions)
Ab8245, by Abcam (2 mentions)
Ab32536, by Abcam (2 mentions)
Image pro plus 6, by Media Cybernetics (2 mentions)
Ab8227, by Abcam (2 mentions)
Ab133462, by Abcam (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Rb 1197 r7, by Thermo Fisher Scientific (2 mentions)
Ab6721, by Abcam (2 mentions)
Ab182734, by Abcam (2 mentions)
Ab194583, by Abcam (2 mentions)
Ab184787, by Abcam (2 mentions)
Ab1416, by Abcam (1 mentions)
Fluoview 300 fluorescence microscope, by Olympus (1 mentions)
Ab260043, by Abcam (1 mentions)
Ab22048, by Abcam (1 mentions)
Ab134047, by Abcam (1 mentions)
Ab16663, by Abcam (1 mentions)
25 protocols using ab194726
1
Immunofluorescence Imaging of Cellular Markers
2
Immunohistochemical Analysis of Liver Inflammation
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The liver specimens were embedded in paraffin and sectioned into 4-μm thick tissue slices. All sections were first stained with hematoxylin and eosin (H&E) for morphological structure analysis. For immunohistochemical staining (IHC), after antigen retrieval and endogenous peroxidase blocking, the sections were incubated with primary antibodies against TLR4 (1/100, 19811-1-AP, Proteintech, Wuhan, China), p-NF-κB p65 (1/50, ab194726, Abcam), IL-6 (use concentration of 1 μg/ml, ab9324, Abcam), and VCAM-1 (1/500, ab134047, Abcam) at 4 °C overnight and then incubated with secondary antibodies at room temperature for 2 h. Freshly prepared 3,3′-diaminobenzidine solution (Boster) was used for coloration. Next, the sections were counterstained with hematoxylin. The brown color indicated positive staining.
3
Immunostaining of Paraffin-Embedded Tissue
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Deparaffinized tissue sections were gradually rehydrated using serial dilutions of ethanol before being boiled for 5 min in sodium citrate buffer (10 mM, pH 6.0), and then processed for immunostaining. The tissue sections were blocked for 2 h in 5% bovine serum albumin (BSA) in tris-buffered saline (TBS). Slides were then incubated for 12 h at 4 °C with primary antibodies against: cyclin-D1 (Catalog # ab16663), IL-6 (Catalog # ab9324), and NF-кB p65 (Catalog # ab194726) (ABCAM, Cambridge, UK). Following a TBS rinse, the sections were incubated with the corresponding secondary antibody. A light microscope was used to visualize the slides and a CDD camera to capture the photomicrographs. Optical density (OD) was quantified using Image J software (1.46 a, NIH, Bethesda, MD, USA) from a minimum of three sections per rat.
4
Molecular Profiling of Neuropathic Pain
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After completing the last behavioral tests at 48 h, all rats were sacrificed
under deep anesthesia (5% sevoflurane). According to the literature,27 (link) the L3-L5
right DRGs were rapidly removed and stored in liquid nitrogen. Tissue samples
were homogenized in lysis buffer, which was centrifuged at 12,000 r/min for
10 min at 4°C to collect the supernatant. The protein concentration was measured
by BCA Protein Assay Kit (Solarbio, Beijing, China). Protein samples were
separated on SDS-polyacrylamide gel electrophoresis, and the fractionated
proteins were transferred to a polyvinylidene difluoride (PVDF) membrane (Thermo
Fisher Scientific, USA). After being blocked with 5% (w/v) skimmed milk for 1 h
at room temperature, the membranes were incubated overnight at 4°C with the
diluted primary antibodies. The primary antibodies used were: anti-TRPA1
(GTX54765, GeneTex, USA), anti-TLR4 (GTX57153, GeneTex, USA), anti- NF-κB
(ab194726, Abcam, USA), and anti-GAPDH (ab8245, Abcam, USA). The membrane was
washed with Tris-buffered saline with Tween buffer and incubated with the goat
anti-mouse secondary antibody (ab150113, Abcam, USA) or goat anti-rabbit
secondary antibody (ab150080 Abcam, USA) for 1 h at room temperature. The bands
were scanned by ECL Western Blotting Substrate (Tanon, China) and analyzed by
Image J software.
under deep anesthesia (5% sevoflurane). According to the literature,27 (link) the L3-L5
right DRGs were rapidly removed and stored in liquid nitrogen. Tissue samples
were homogenized in lysis buffer, which was centrifuged at 12,000 r/min for
10 min at 4°C to collect the supernatant. The protein concentration was measured
by BCA Protein Assay Kit (Solarbio, Beijing, China). Protein samples were
separated on SDS-polyacrylamide gel electrophoresis, and the fractionated
proteins were transferred to a polyvinylidene difluoride (PVDF) membrane (Thermo
Fisher Scientific, USA). After being blocked with 5% (w/v) skimmed milk for 1 h
at room temperature, the membranes were incubated overnight at 4°C with the
diluted primary antibodies. The primary antibodies used were: anti-TRPA1
(GTX54765, GeneTex, USA), anti-TLR4 (GTX57153, GeneTex, USA), anti- NF-κB
(ab194726, Abcam, USA), and anti-GAPDH (ab8245, Abcam, USA). The membrane was
washed with Tris-buffered saline with Tween buffer and incubated with the goat
anti-mouse secondary antibody (ab150113, Abcam, USA) or goat anti-rabbit
secondary antibody (ab150080 Abcam, USA) for 1 h at room temperature. The bands
were scanned by ECL Western Blotting Substrate (Tanon, China) and analyzed by
Image J software.
5
TLR4/MyD88/NF-kB Signaling Pathway Analysis
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The ileum tissues were lysed in RIPA buffer (Cat No. P0013C; Beyotime) and the total protein concentrations were detected through a BCA kit (Cat No. P0012S; Beyotime). Immunoblotting was carried out as previously reported. Proteins (50 μg/lane) were separated by 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis gels. After blocking the PVDF membrane (Cat No. IPVH00010; Millipore, USA), they were subsequently incubated overnight with primary antibodies against TLR4 (dilution ratio 1:1,000, AF7017; Affinity, China), MyD88 (dilution ratio 1:500, AF5192; Affinity), p-NF-kB (dilution ratio 1:1,000, ab194726; Abcam), NF-kB (dilution ratio 1:1,000, ab16502; Abcam), and GAPDH (dilution ratio 1:3,000, AF7021; Affinity). Then, the membranes were incubated with secondary HRP-conjugated antibodies (dilution ratio 1:3,000, S0001; Affinity) at room temperature for 2 h. The immunoreactive bands were then analyzed using the Western ECL reagent (Cat No. RPN2106V1, GE Healthcare, UK) according to the protocol of the manufacturer.
6
Comprehensive Protein Expression Analysis
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Western blot analysis was carried out on whole cell lysates. Antibodies against Tim-3 (Ab241332), NF-κB (p65) (Ab16502), p-NF-κB (p-p65) (Ab194726) and vascular endothelial growth factor (VEGF) A (Ab9570) were purchased from (Abcam Cambridge, UK). GAPDH (sc-47724), p-β-catenin (sc-16743-R), cyclin D1 (CCND1) (sc-8396), C-Myc (sc-70465), matrix metalloproteinase-1 (MMP1) (sc-21731), TWIST (sc-6269), zona occludens (ZO)-1 (sc-10804), ZO-2 (sc-11448), occludin (sc-133256) and VEGFB (sc-13083) antibodies were obtained from Santa Cruz (Insight Biotechnology Limited, Middlesex UK). E-Cadherin (AF748) and VEGFD (MAB286) antibodies were purchased from R&D Systems (Abingdon, Oxfordshire, UK). STAT3 (S5933), p-STAT3 (SAB4504541), interleukin (IL)-6 (17901) and β-catenin (C2206) antibodies were purchased from Sigma-Aldrich (Gillingham, Dorset, UK). Anti-mouse (A5278), anti-rabbit (A0545) and anti-goat (A8919) secondary antibodies were obtained from Sigma-Aldrich (Gillingham, Dorset, UK).
7
Antibody Production and Validation for Shrimp
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The polyclonal antibodies for L. vannamei Dorsal and Cactus were produced in guinea pigs and rabbits, respectively, by GL Biochem antibody manufacturing company (China) from our previous study [49 (link)]. Polyclonal rabbit anti-NF-κB p65 (phospho S276) antibody (ab194726, Abcam) was used to detect the phosphorylated shrimp Dorsal [51 (link)]. Rabbit anti-Histone H3 (4499s), Rabbit anti-Hsp90 (ab13495), and the secondary antibodies Goat Anti-Guinea pig IgG H&L (Alexa Fluor 488) (ab150185), Goat anti-Guinea pig IgG H&L (HRP) (ab6908), anti-Mouse IgG H&L (HRP) (ab6789) and anti-Rabbit IgG H&L (HRP) (ab6721), were purchased from Abcam (USA). Mouse anti-Actin antibody was obtained from Merck (MAB1501). Mouse anti-6His antibody (H1029) and Mouse anti-GST antibody (SAB4200237) were gained from Sigma (USA).
8
Protein Extraction and Western Blot Analysis
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Cells were trypsinized, pelleted and washed twice with cold phosphate-buffered saline (PBS). Cell pellets were then lysed in cold low-ionic-strength buffer (50 mM NaCl, 1% IGEPAL, 10% glycerol, 10 mM HEPES, pH 7.4) with fresh added proteinase inhibitor cocktail, DTT and PMSF (ThermoFisher Scientific, San Jose, CA, USA). After the determination of protein concentration, samples were denatured in SDS sample buffer, sonicated three times for 15 s each time (Branson150) and centrifuged at 10,000 rpm for 20 min. Supernatants were heated at 95 °C for 5 min. Proteins were resolved using Criterion TGX 4–15% precast PAGE gels and transferred to nitrocellulose membranes with a Bio-Rad Trans-Blot Turbo transfer system. Primary antibodies used were anti-RelA (ab194726, Abcam, Waltham, MA, USA; MAB5078, R&D, Minneapolis, MN, USA); anti-actin (937215, R&D) was used as a loading control. Blots were imaged and quantified.
9
Immunoblotting Assay for Apoptosis Markers
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The protein levels of cyclin D1, ki67, caspase-8, caspase-3, cleaved-caspase-8, cleaved-caspase-3, RIPK1, IKKβ, p-IκBα, and p-p65 were examined by immunoblotting following the methods described before (36 (link)) with antibodies against cyclin D1 (60186-1-Ig; Proteintech, Wuhan, China), ki67 (27309-1-AP, Proteintech), caspase3 (19677-1-AP, Proteintech), cleaved-caspase 3 (ab2302, Abcam, Cambridge, MA, USA), caspase-8 (ab32397, Abcam), cleaved-caspase-8 (# 9496S; Cell Signaling; Danvers, MA, USA), IKKβ (07-1008; Sigma-Aldrich, St. Louis, MO, USA), p-IκBα (#9246; CST, Danvers, MA, USA), p-p65 (ab194726, Abcam), and β-actin (60008-1-Ig, Proteintech). β-actin was taken as an endogenous control. The incubation of membranes with primary antibody was followed by another incubation with HRP-conjugated secondary antibodies. Protein blots were then visualized using enhanced chemilumescent (ECL) substrates (Millipore, MA, USA).
10
Western Blot Analysis of Apoptosis and Oxidative Stress Markers
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Cells were lysed in RIPA buffer (Cell Signaling Technology), and equivalent amounts of protein extracts were separated using 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). Protein samples were then transferred to polyvinylidene fluoride membranes and blocked with 5% non-fat milk. These membranes were then incubated with primary antibodies, including rabbit anti-Bcl-2 (1 : 2000, ab182858, Abcam), rabbit anti-Bax (1 : 1000, ab32503, Abcam), rabbit anti-c-caspase3 (1 : 500, ab13847, Abcam), rabbit anti-caspase 3 (1 : 5000, ab32351, Abcam), rabbit anti-NOX4 (1 : 2000, ab133303, Abcam), rabbit NF-κB p65 (1 : 2000, ab32536, Abcam), rabbit p-NF-κB p65 (1 : 1000, ab194726, Abcam), and rabbit anti-β-actin (1 : 200; ab115777, Abcam) overnight at 4°C. Afterward, the membranes were gently washed and then incubated with horseradish peroxidase-labelled secondary antibody (goat anti-rabbit, 1 : 2000, ab205718, Abcam) at 25°C for 1 hour. Finally, relative expression level was quantified using a Gel-Pro Analyzer (Media Cybernetics) and normalized to β-actin.
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