Sections (4 μm thick) from the paraffin-embedded colon tissue blocks were prepared for IHC or IF staining. Briefly, staining was performed with primary antibodies against TNF-α (1 : 600, rabbit polyclonal, Bioss, bs-2081R), IL-1β (1 : 2,000, rabbit polyclonal, ABclonal, A11369), NF-κB/p65 (1 : 5,000, rabbit polyclonal, Servicebio, GB11142), F4/80 (1 : 1000, CST, 70076), CD86 (1 : 500, Bioss, bs-1035R), and CD163 (1 : 300, Bioss, bs-2527R). Goat anti-rabbit IgG (HRP) (1 : 4,000, Abcam, ab205718), Alexa 488-conjugated donkey anti-rabbit IgG(H+L) (1 : 400, Life Technologies, A21206), and Cy3 (1 : 200) were used as secondary antibody staining reagents for IF staining, while a goat polyclonal secondary antibody recognizing mouse IgG (1 : 500, Abcam, ab150113) was used for immunohistochemistry; DAPI-containing TBST was applied for 5 min. Sections were visualized using an Olympus microscope (Olympus BX53, Olympus Corporation).
Bs 2081r
Manufactured by Bioss Antibodies
Sourced in China, United States
The Bs-2081R is a laboratory equipment product manufactured by Bioss Antibodies. It is a device designed for general-purpose laboratory applications. The core function of the Bs-2081R is to perform tasks commonly associated with typical lab equipment, though a detailed description is not available while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
Bs 1035r, by Bioss Antibodies (2 mentions)
A21206, by Thermo Fisher Scientific (1 mentions)
Ab150113, by Abcam (1 mentions)
Bs 2527r, by Bioss Antibodies (1 mentions)
Ab205718, by Abcam (1 mentions)
Ab23990, by Abcam (1 mentions)
Ab157210, by Abcam (1 mentions)
Ab134189, by Abcam (1 mentions)
Ab9324, by Abcam (1 mentions)
Bs 2211r, by Bioss Antibodies (1 mentions)
Bs 0698r, by Bioss Antibodies (1 mentions)
Bm0627, by Boster Bio (1 mentions)
Bs 10011r, by Bioss Antibodies (1 mentions)
Spss statistical software package version 25, by IBM (1 mentions)
Bs 6319r, by Bioss Antibodies (1 mentions)
Bs 0279r, by Bioss Antibodies (1 mentions)
Rabbit anti iba1, by Fujifilm (1 mentions)
Evos fl auto imaging system, by Thermo Fisher Scientific (1 mentions)
Ab225844, by Abcam (1 mentions)
Ab172967, by Abcam (1 mentions)
9 protocols using bs 2081r
1
Immunostaining of Colon Tissue
2
Comprehensive Protein Expression Analysis
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A total cell protein extraction kit (Milipore, Billerica, MA, USA) was used to extract total protein. An equivalent amount of protein from each sample was electrophoresed by 12% SDS-PAGE and transferred to nitrocellulose membrane. After being blocked, membranes were incubated with anti-TLR4 (1:1000; PA5-23124, Invitrogen), anti- MHC-I (1:1000; ab134189 and ab22367, Abcam), anti-MHC-II (1:1000; ab157210 and ab23990, Abcam), anti-CD80 (1:1000; PA5-19211, Invitrogen and bs-2211r, Bioss, Woburn, MA, USA), anti-CD86 (1:1000; bs-1035r, Bioss), anti-TNF-α (1:1000; bs-2081R, Bioss), anti-IL-6 (1:2000; ab9324, Abcam), anti-IL-10 (1:1000; bs-0698r, Bioss), anti-CXCL10 (1:1000; PAA371Ra01; Cloud-Clone Corp., Houston, TX, USA), anti-TGF-β1 (1:1000; c0340, Assay biotechnology, Sunnyvale, CA, USA) and TGF-β2 (1:1000; 5343r-100, BioVision, Milpitas, CA, USA) overnight at 4 °C. Membranes were then washed three times with PBS/0.1%Tween-20 (5 min each), and incubated with a corresponding secondary antibody (1:5000) for 2 h at room temperature. Bands were detected using a chemiluminescence ECL kit (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and were quantified using the Sigma-Gel software (Jandel Scientific Software, Sari Kafael, CA, USA).
3
Western Blot Analysis of Inflammatory Markers
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Standard WB procedures were carried out with those antibodies: rabbit anti-TNF alpha polyclonal antibody (bs-2081R, bioss, Beijing, China), IL10 antibody (GTX632359, Gene Tex, CA, USA), rabbit anti-occludin polyclonal antibody (bioss, bs-10011R), anti-NF-κB (CST, mAb #8242, Danvers, MA, USA), TLR4 antibody (NB100-56566SS, Novus, CO, USA), mouse anti-β-actin (BM0627, boster, Wuhan, China).
4
Immunohistochemical Analysis of TNF-α and IL-1β in Rat Eye Tissue
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TNF-α and IL-1β were detected in the rat eye tissue with EIU by immunohistochemical staining using rabbit polyclonal antibodies (TNF-α;bs-2081R, IL-1β;bs-6319R, Bioss, USA) and the streptavidin–biotin peroxidase technique. The procedure was performed under the same conditions for all sections as previously described.[13 (link)]
Immunohistochemical evaluations were performed using the extensity of the staining. The distribution (0.1: <25%, 0.4: 26%–50%, 0.6: 51%–75%, 0.9: 76%–100%) and intensity (0: no staining; +0.5: very little staining; +1: little staining; +2: medium staining; +3: very strong staining) of immune reactivity was used to obtain a histoscore (Histoscore = distribution × intensity).[14 (link)]
The SPSS statistical software package version 25.0 (SPSS Inc., Chicago, IL, USA) was used to analyze the data. The data were reported as mean ± standard deviations for each data set. A statistical significance was considered if P < 0.05. The statistical analyses of the data were performed using one-way analysis of variance test, Kruskal–Wallis test and post hoc analyses (Tukey test and pairwise comparisons using Bonferroni correction, respectively).
Immunohistochemical evaluations were performed using the extensity of the staining. The distribution (0.1: <25%, 0.4: 26%–50%, 0.6: 51%–75%, 0.9: 76%–100%) and intensity (0: no staining; +0.5: very little staining; +1: little staining; +2: medium staining; +3: very strong staining) of immune reactivity was used to obtain a histoscore (Histoscore = distribution × intensity).[14 (link)]
The SPSS statistical software package version 25.0 (SPSS Inc., Chicago, IL, USA) was used to analyze the data. The data were reported as mean ± standard deviations for each data set. A statistical significance was considered if P < 0.05. The statistical analyses of the data were performed using one-way analysis of variance test, Kruskal–Wallis test and post hoc analyses (Tukey test and pairwise comparisons using Bonferroni correction, respectively).
5
Immunohistochemical Analysis of VEGF and TNF-α in Rat OIR
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VEGF and TNF-α were detected in the rat eye tissue with OIR by immunohistochemical staining using rabbit polyclonal antibodies (VEGF; bs-0279R, TNF-α; bs-2081R, Bioss, USA) and the streptavidin–biotin peroxidase technique. The procedure was performed as described previously[27 (link)] and identical conditions were applied for all tissue sections.
Extensity of the staining was taken as the basis for evaluation of immunohistochemical staining. A histoscore was calculated by multiplying the distribution (0.1: <25%, 0.4: 26%–50%, 0.6: 51%–75%, 0.9: 76%–100%) by intensity (0: no staining; +0.5: very little staining; +1: little staining; +2: medium; +3: very strong) to evaluate the immune reactivity (Histoscore = distribution × intensity).[28 (link)]
Extensity of the staining was taken as the basis for evaluation of immunohistochemical staining. A histoscore was calculated by multiplying the distribution (0.1: <25%, 0.4: 26%–50%, 0.6: 51%–75%, 0.9: 76%–100%) by intensity (0: no staining; +0.5: very little staining; +1: little staining; +2: medium; +3: very strong) to evaluate the immune reactivity (Histoscore = distribution × intensity).[28 (link)]
6
Immunostaining and Western Blot for Microglia
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The antibodies used for immunostaining were as follows:
Rabbit anti-Iba-1(Iba-1 as microglial marker, 1:400,019-19741, Wako Chemicals, VA, USA);
Rabbit anti-TNF-α (1:500, bs-2081R, Bioss, Beijing, China);
7
Histological Analysis of Tissue Samples
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The paraffin-embedded tissues were sliced and performed with hematoxylin-eosin (HE), Sirius red, or Masson trichrome staining. The interstitial fields and the perivascular fields containing vasculature were imaged. Immunohistochemistry was performed with antibodies of IL-6 (D220828, Sangon, Shanghai, China), TNF-α (bs-2081R, Bioss, Beijing, China), and ANP and MYH7B (ab225844 and ab172967, Abcam, U.S.). The EVOS FL Auto Imaging System (Life Technologies, ThermoFisher Scientific, Waltham, MA, U.S.) was operated to obtain 3-5 random fields of each image.
8
Immunohistochemical Analysis of Skin Samples
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Immunohistochemical analysis of skin samples from one of the participants (case 1) was performed as described previously (17 (link)), with slight modifications. Thin sections (4 μm) were cut from samples embedded in paraffin blocks. The sections were soaked for 20 min at room temperature in 0.3% H2O2/methanol to block endogenous peroxidase activity. After being washed in PBS with 0.01% Triton X-100, the sections were incubated for 30 min in PBS with 4% bovine serum albumin, followed by incubation overnight with the primary antibodies, polyclonal rabbit anti-IL-17C antibody (bs-2611R, Bioss, Woburn, MA; dilution 1:1000), anti-IL-36γ antibody (ab156783; Abcam, Cambridge, UK; dilution 1:1000), and anti-TNF-α antibody (bs-2081R; Bioss; dilution 1:1000) in PBS containing 1% bovine serum albumin. After being washed in PBS, the thin sections were stained with Dako EnVision+Single Reagents (HRP, rabbit) (Agilent Technologies, Santa Clara, CA) for 30 min at room temperature. An Olympus BX51 (Olympus Corporation, Tokyo, Japan) was used for photography.
9
Immunohistochemical Analysis of TNFα and Caspase-3
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For immunohistochemical analysis, sections were mounted on polylysine coated slides. After rehydrating, samples were transferred to citrate buffer (pH 7.6) and heated in a microwave oven for 20 minutes. After cooling for 20 minutes at room temperature, the sections were washed with phosphate buffered saline (PBS). Then sections were kept in 0.3% H 2 O 2 for 7 minutes and afterward washed with PBS. Sections were incubated with at an anti-TNFα (1:100, bs-2081R, Bioss, China) and anti-Caspase 3 (1:100, bs-0081R, Bioss, China) for 60 minutes. They then were rinsed in PBS and incubated with biotinylated goat antipolyvalent for 10 minutes and streptavidin peroxidase (SHP 125, ScyTek Laboratories, ABD) for 10 minutes at room temperature. Staining was completed with chromogen + substrate for 15 minutes, and slides were counterstained with Mayer's hematoxylin (M06002, Bio-optica, ITALY) for 1 minute, rinsed in tap water, and dehydrated. The antibody was used according to the manufacturer's instructions. Staining for anti-TNFα and anti-caspase-3 was identi ed by a brown color. The relative intensity of TNFα and caspase-3 immunostaining was scored as follows: 0-5% (+), 6-20% (++), 21-40% (+++), 41-60% (++++), 61-80% (+++++), and 81-100% (++++++). All sections were examined using a Nikon Eclipse 80i light microscope and Nikon Image Analysis system.
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