The immunohistochemistry in this experiment included the following antibodies: caspase-3 (CASP3; GB11009-1; Servicebio, Wuhan, China; 9 rats from each of the CA and CM groups); interleukin-6 (IL-6; GB11117; Servicebio; 13 and 14 rats from the CA group and CM groups, respectively); mammalian target of rapamycin (mTOR; GB11117; Servicebio; 13 rats from each of the CA and CM groups); nuclear factor-kappa B (NF-κB; 13,533–1- AP; Proteintech, Wuhan, China; 9 rats from each of the CA and CM groups); and tumor necrosis factor-alpha (TNF-α; bsm-33207 m; Bioss, Beijing, China; 13 and 14 rats from the CA group and CM groups, respectively). The experimental method of this part is in the Additional file 3 : Experimental methods.
Gb11117
Manufactured by Wuhan Servicebio Technology
Sourced in China
The GB11117 is a laboratory equipment product. It serves as a core function for various laboratory applications. No further details are available on the specific intended use or capabilities of this product.
Automatically generated - may contain errors
Lab products found in correlation
Gb11188, by Wuhan Servicebio Technology (5 mentions)
Primo star, by Zeiss (2 mentions)
Af5103, by Wuhan Servicebio Technology (2 mentions)
Gb11499, by Wuhan Servicebio Technology (2 mentions)
Gb23303, by Wuhan Servicebio Technology (2 mentions)
Sc 57342, by Santa Cruz Biotechnology (1 mentions)
A32728, by Thermo Fisher Scientific (1 mentions)
Yp0575, by Immunoway (1 mentions)
A10521, by Thermo Fisher Scientific (1 mentions)
A10520, by Thermo Fisher Scientific (1 mentions)
Ab14106, by Abcam (1 mentions)
Ab32570, by Abcam (1 mentions)
Cw0103, by CWBIO (1 mentions)
Ab5694, by Abcam (1 mentions)
Dab reagent, by Boster Bio (1 mentions)
Gb12001, by Wuhan Servicebio Technology (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Phenylmethylsulfonyl fluoride, by Beyotime (1 mentions)
Il 1β, by Affinity Biosciences (1 mentions)
Gb111629, by Wuhan Servicebio Technology (1 mentions)
14 protocols using gb11117
1
Immunohistochemical Analysis of Signaling Pathways
2
Multicolor Immunofluorescent Profiling of LDLR, InsR, and p-AMPK
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Triple immunofluorescent staining of LDLR, InsR, and p-AMPK in hepG2 cells and liver tissue, was conducted by incubating simultaneously with a mouse InsR monoclonal antibody (1:50, sc-57342, Santa Cruz, USA), rabbit polyclonal antibody against p(Thr183/172)-AMPKα1/2 (1:100, YP0575, ImmunoWay, USA) and Alexa Fluor 488-conjugated rabbit LDLR antibody (1:100, ab196377, Abcam, UK). Then, samples were stained with CY3-conjugated goat anti-mouse secondary antibody (1:200, A10521, Thermofisher, USA) and Alexa Fluor 647-conjugated goat anti-rabbit secondary antibody (1:200, A32728, Thermofisher, USA). For double staining of IL-6 and TNF-α in liver and epididymal fat tissues, samples were stained with rabbit IL-6 (1:100, GB11117, Servicebio, China) and mouse TNF-α (1:100, GB11188, Servicebio, China) primary antibody and then incubated with FITC-conjugated goat anti-mouse secondary antibody (1:100, A16079, Thermo Fisher, USA) and CY3-conjugated goat anti-rabbit secondary antibody (1:100, A10520, Thermo Fisher, USA). Finally, all samples were mounted with DAPI. Fluorescence pictures were photographed on a C2t Nikon fluorescent microscope. The excitation at 377 nm was for nucleus, 494 nm for LDLR and TNF-α, 543 nm for InsR and IL-6, 628 nm for p-AMPK. The emission was collected at 447 nm for nucleus, 527 nm for LDLR and TNF-α, 586 nm for InsR and IL-6, 690 nm for p-AMPK.
3
IHC Staining of Bladder Tissue Markers
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Previously described standard procedures were used for IHC [30 (link), 31 (link)]. The primary antibodies used were TAGLN (1:200, #ab14106; Abcam), α-SMA (1:200, #ab5694; Abcam), PDGFR-β (1:200, #ab32570; Abcam,), and IL-6 (1:200, #GB11117; Servicebio). The secondary antibody used for all IHC procedures was the horseradish peroxidase‑conjugated goat anti-rabbit (#CW0103S; CWBIO). For the TAGLN IHC expression analysis, normal mouse bladder tissues with and without primary antibody were used as positive and negative controls, respectively (Supplementary Fig. 1B ).
4
Western Blot Protocol for Protein Analysis
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Western blot analysis was performed using standard procedures, as described previously [61 (link)]. To obtain proteins, cells were lysed by 100 mg/mL RIPA lysis buffer containing 1% phenylmethylsulfonyl fluoride (Beyotime, Shanghai, China) on ice. Then, cell lysates were boiled in 5× SDS buffer for 5 min before being separated by 10% SDS-polyacrylamide gel, and transferred to a PVDF membrane (Bio-rad, Hercules, CA, USA). Subsequently, the membranes were blocked by TBST containing 5% non-fat dried milk for 2 h at 37 °C to avoid non-specific binding. After that, the membranes were incubated with primary antibodies overnight at 4 °C. Additionally, the immunoblot membranes were incubated with the secondary antibodies for 2 h at 37 °C after washing 3 times. The blots were visualized by DAB reagent (Boster, Wuhan, China) according to the manufacturer’s instructions. The antibodies included anti-β-actin (GB12001, Servicebio, Wuhan, China, dilution: 1:1000); anti-IL6 (GB11117, Servicebio, China, dilution: 1:1000); anti- IL 1 β (abs115412, Absin, Shanghai, China, dilution: 1:1000).
5
Immunohistochemical Analysis of Inflammatory Markers and Cell Proliferation
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To demonstrate the proliferation of epidermal cells and detect the inflammatory response, immunohistochemistry stain was performed according to a previous study [26 (link)]. The sliced tissues were incubated with primary antibodies, including rabbit anti-mouse interleukin-1β (IL-1β, Affinity; AF5103, China; 1:100), interleukin-6 (IL-6, Servicebio; GB11117, China; 1:600), tumor necrosis factor α (TNF-α, Servicebio; GB11188, China; 1:500), and Ki67 (Servicebio; GB11499, China; 1:500) at 4 °C for 12 h. Secondary antibodies (Servicebio; GB23303, China; 1:400) were used for incubation at 37 °C for 1 h, and diaminobenzidine staining (Biosharp, China) was performed. The positive staining of IL-1β, IL-6, TNF-α, and Ki67 was recorded by light microscopy (Primo Star, Zeiss, Germany) and measured by ImageJ software.
6
Bladder Tissue Immunostaining Analysis
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Bladder sections were prepared as described above. Slides were blocked and incubated with anti-ki-67 (1:100, GB111141, Servicebio, China), anti-IL-6 (1:100, GB11117, Servicebio, China), anti-Collagen I (1:100, 14695-1-AP, Proteintech, China), and anti-Collagen III (1:100, GB111629, Servicebio, China). After the slides were incubated with relevant secondary antibodies, five images were randomly photographed and recorded using a BX53F fluorescence microscope (Olympus, Tokyo, Japan). Image-Pro Plus was employed to calculate the ratio between positive areas and the total area for semi-quantitative analysis.
7
Histological Evaluation of Allograft Rejection
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Allografts were harvested at POD 7 or at the time of complete cessation of heart beats. The tissues were fixed in formalin and embedded in paraffin. The paraffin-embedded tissue sections were stained with hematoxylin and eosin (H&E) staining, and the grade of acute rejection was assessed according to the criteria of ISHLT (Stewart et al., 2005 (link)). T lymphocyte infiltration of the tissues was determined by immunohistochemical staining with anti-CD3 antibody (ab5690, Abcam, Cambridge, UK). The secretion of inflammatory cytokines was determined by immunofluorescence with anti-IFN-γ (ab216642, Abcam, Cambridge, UK), anti-IL-2 antibody (ZI091710A, R&D, Minneapolis, MN) and anti-IL-6 (GB11117, Servicebio, Wuhan, China). Image-J software was used to quantify fluorescence images (Bethesda, MD).
8
Immunohistochemical Analysis of Skin Regeneration
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Immunohistochemical staining was performed to investigate epidermal cell proliferation and inflammatory factors expression in regenerating skin tissue, as per previous study [46 (link)]. Primary antibodies, including rabbit anti-mouse IL-1β (Affinity, af5103, China, 1:100), IL-6 (Servicebio, gb11117, China, 1:600), TNF-α (Servicebio, gb11188, China, 1:500), and Ki67 (Servicebio, gb11499, China, 1:500) were used following the provided instructions. After incubation, the skin wound tissue was incubated with secondary antibodies (Servicebio, GB23303, China, 1:400) for 1 h at 37 °C, then sealed and observed by light microscopy (Primo Star, Zeiss, Germany). ImageJ software was used to measure positive staining intensity and analyzed by GraphPad Prism software.
9
Histological Analysis of Osteoclast Formation
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After the micro-CT analysis was completed, decalcification was carried out in 10% ethylenediaminetetraacetic acid solution at 4°C for 4 days, followed by paraffin embedding. Histological sections (5 mm thick) were cut and prepared in the coronal plane for tartrate-resistant acid phosphatase (TRAP) staining, hematoxylin and eosin (H&E) staining, and detection of TNF-α (GB11188, Servicebio, China) and IL-6 (GB11117, Servicebio, China). Detection of SPHKs, BECN1, and TRAF2 was performed on the specimens. The number of TRAP-positive multinucleated OCs was assessed in each calvaria and specimens. Stained sections were observed and photographed under an optical microscope (Leica Aperio CS2, Germany). The Image-Pro Plus 6.0 software was used to analyze the ROI of each section.
10
Immunohistochemical Profiling of Macrophage Subsets
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Pancreatic sections were deparaffinized and rehydrated. Tris-EDTA (pH 9.0, G1203, Servicebio, China) was used for antigen retrieval, 0.2% Triton X-100 was used for permeabilization, and 10% donkey serum was used for blocking. The sections were incubated with primary antibodies overnight at 4°C and incubated with secondary antibodies for 1 h at 37°C. For immunofluorescent staining, primary macrophages were fixed in 4% paraformaldehyde for 20 min and blocked in 10% donkey serum. Incubation with primary antibodies was performed overnight at 4°C. Secondary antibody incubation was performed for 1 h at 37°C and DAPI (ab104139, Abcam, U.S.A.) was used to label the nuclei. The primary antibodies included CD68 (1:50, Sc-20060, Santa Cruz), iNOS (1:200, 18985-1-AP, Proteintech), CD206 (1:200, 18704-1-AP, Proteintech), IL-6 (1:200, GB11117, Servicebio), and Hes1 (1:200, ab108937, Abcam). The secondary antibodies included donkey anti-rabbit IgG H&L (Alexa Fluor® 488) (1:200, ab150073, Abcam), and donkey anti-mouse IgG H&L (Alexa Fluor® 594) (1:200, ab150108, Abcam). Five sections in each group were examined and ten 400× images per section were evaluated. iNOS+CD68+ cells represented M1 macrophages, CD206+CD68+ cells represented M2 macrophages, and Hes1+CD68+ cells represented Hes1-positive macrophages.
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