As previously described [15 (link)], immunohistochemistry was performed on formalin-fixed 3-μm-thick paraffin-embedded sections. Briefly, sections were deparaffinized, rehydrated using an ethanol series, blocked with normal horse serum, treated with primary antibodies at 4°C overnight, and incubated for 30 minutes with a secondary antibody (ImmPRESS HRP reagent kit, Vector Laboratories, Burlingame, CA, USA). The slides were developed using 3,3′-diaminobenzidine (DAB; Vector Laboratories) and counterstained with Harris hematoxylin. The primary antibodies used were anti-phosphorylated Akt1 (p-Akt1) (ab5954, 1:200, Abcam, Cambridge, UK), anti-E-cadherin (#3195, 1:400, Cell Signaling Technology, Danvers, MA, USA), anti-transforming growth factor-β1 (TGF-β1) (ab190503, 1:500, Abcam), anti-phosphorylated glycogen synthase kinase-3β (p-GSK-3β) (ab131097, 1:100 dilution, Abcam), anti-β-catenin (#9562, 1:400, Cell Signaling Technology), and anti-Snail (sc-271977, 1:100, Santa Cruz Biotechnology, Santa Cruz, CA, USA). Staining intensity was semi-quantitatively graded in a blinded manner using the following scoring scale: 0 = absence of specific staining; 1, < 25% area with specific staining; 2, 25% to 50% staining; 3, 50% to 75% staining; 4, > 75% staining. At least 20 fields of renal cortex were investigated [16 (link)].
Ab190503
Manufactured by Abcam
Sourced in United Kingdom
Ab190503 is an Enzyme-linked immunosorbent assay (ELISA) kit designed for the quantitative measurement of human Interferon gamma (IFN-γ) in cell culture supernatants, serum, and plasma samples. The kit utilizes the sandwich ELISA technique and includes all the necessary reagents for performing the assay.
Automatically generated - may contain errors
Lab products found in correlation
Ab131097, by Abcam (1 mentions)
Anti e cadherin, by Cell Signaling Technology (1 mentions)
Immpress hrp reagent kit, by Vector Laboratories (1 mentions)
Ab175394, by Abcam (1 mentions)
Ab993, by Abcam (1 mentions)
Enhanced chemiluminescence reagent, by Bio-Rad (1 mentions)
Ab179475, by Abcam (1 mentions)
Goat anti rabbit secondary antibody, by Bio-Rad (1 mentions)
Horseradish peroxidase conjugated anti mouse igg, by Cell Signaling Technology (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
Sds page gel, by Bio-Rad (1 mentions)
Ab202445, by Abcam (1 mentions)
Ab63399, by Abcam (1 mentions)
Chemidoc xrs system, by Bio-Rad (1 mentions)
Ab32064, by Abcam (1 mentions)
Gtx61206, by GeneTex (1 mentions)
Gtx83114, by GeneTex (1 mentions)
Ab32518, by Abcam (1 mentions)
Ab193297, by Abcam (1 mentions)
Lps escherichia coli o111 b4, by Merck Group (1 mentions)
5 protocols using ab190503
1
Immunohistochemical Analysis of Renal Biomarkers
2
Western Blot Analysis of Protein Expression
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30μG of total protein was separated by SDS-PAGE gel (4–20% resolving; Bio-Rad Laboratories) and transferred to a PDVF membrane. Non-specific binding was blocked by 5% BSA in tris-buffered saline for 1 h at room temperature prior to overnight incubation with primary antibody at 4 °C. Membrane was then washed and incubated with horseradish peroxidase-conjugated anti-mouse IgG (1:4000; Cell Signaling Technologies, Danvers, MA, USA, #7076), or goat anti-rabbit secondary antibody (1:4000; Bio-Rad Laboratories, #1706515) for 2 h at room temperature. Protein bands were visualized using the enhanced chemiluminescence reagents (Bio-Rad Laboratories). Quantification by densitometry with Image Lab™ software (Bio-Rad Laboratories) was performed using β-actin as internal control. To probe for respective proteins, we used anti-ITGAV (1:2500; Abcam, Cambridge, UK; ab179475), anti-AKR1B1 (1:1000; Abcam, ab175394), anti-TGFβ1 (1:1000; Abcam, ab190503), anti-G6PD (1:5000, Abcam, ab993) and anti-actin (1:1000; Cell Signaling Technologies, #8457) antibodies.
3
TGF-β1 and Smad2/3 Signaling Analysis
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Total proteins were extracted from the LV and RV and protein concentrations were determined through the bicinchoninic acid method. The primary antibodies were as follows: anti-TGF-β1 monoclonal antibody (1:500; Abcam, ab190503, Cambridge, United Kingdom), anti-Smad2/3 monoclonal antibody (1:1,000; Abcam, ab202445, Cambridge, United Kingdom), and anti-pSmad2/3 polyclonal antibody (1:500; Abcam, ab63399, Cambridge, United Kingdom). Secondary antibodies were goat anti-rabbit and goat anti-mouse (KPL, Milford, MA, United States). The images were captured and semi-quantitatively analyzed by the ChemiDoc XRS + System (Bio-Rad, Hercules, CA, United States).
4
Immunohistochemical Analysis of Protein Markers
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Tissues were embedded in paraffin and sliced for IHC experiment to verify the protein expression. The antibodies against Ki67 (550609, 1:50, BD Biosciences); PARP (ab32064, 1:100, Abcam); fibronectin (GTX61206, 1:250, GeneTex); MMP-9 (10375-2-AP, 1:250, Proteintech); TGF-β (ab190503, 1:100, Abcam); vimentin (550513, 1:50, BD Biosciences) and CD44 (GTX83114, 1:500, GeneTex) were used.
5
Moracin Modulates Inflammatory Response
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The drug, moracin (wkq-00871) was purchased from Sichuan Victory Biological Technology Co., Ltd (Chengdu, China). LPS (Escherichia coli O111:B4) was purchased from Sigma (St. Louis, MO, U.S.A.). Catalase (CAT, LE-06378), malondialdehyde (MDA, LE-07345) and superoxide dismutase (SOD, LE-07334) detection kits were bought from Lai Er Bio-Tech (Hefei, China). Enzyme-linked immunosorbent assay (ELISA) kits for IL-6 (CRC0063), TNF-α (BMS622) and IL-1β (BMS630) were obtained from eBioscience. CO., LTD. All the primary antibodies used in the present study, including antibodies for Nrf-2 (ab137550), HO-1 (ab13243), TGF-β (ab190503), p-smad-3 (ab193297), smad-3 (ab40854), p-IκBα (ab133462), IκBα (ab32518), p-NF-κBp65 (ab86299), NF-κBp65 (ab16502) were from Abcam (Cambridge, U.K.).
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