After preservation, the kidney sections were boiled for antigen retrieval and treated with 3% H2O2 to remove endogenous peroxidases. After rinsing with PBS, goat serum was used to block the sections. The sections were incubated at 4°C overnight with a polyclonal OPN antibody (rabbit polyclonal to OPN, ab8448, Abcam, Cambridge, UK) (5 μg/mL) or a polyclonal KIM-1 antibody (rabbit polyclonal to KIM-1, Hopebiot) (1 : 500). After rinsing with PBS, the sections were incubated at 37°C with a polymer helper for 20 min. After rinsing three times with PBS, the slides were treated with poly-peroxidase-anti-rabbit/mouse IgG for 2 h before a final wash with PBS and staining with DAB.
Kim 1
Manufactured by Abcam
Sourced in United States, United Kingdom
KIM-1 is a lab equipment product that functions as a biomarker for kidney injury. It is a protein that is expressed in the kidneys and can be detected in the blood and urine of individuals with kidney damage. KIM-1 plays a role in the regeneration and repair of the kidney following injury.
Automatically generated - may contain errors
Lab products found in correlation
Bcl 2, by Abcam (5 mentions)
Collagen 1, by Abcam (4 mentions)
Il 1β, by Abcam (4 mentions)
β actin, by Abcam (4 mentions)
Bcl2 associated x (bax), by Abcam (3 mentions)
Galectin 3, by Abcam (2 mentions)
4 hydroxynonenal 4 hne, by Abcam (2 mentions)
Cleaved caspase 3, by Abcam (2 mentions)
β actin, by Cell Signaling Technology (2 mentions)
α sma, by Abcam (2 mentions)
Vimentin, by Abcam (2 mentions)
Il 18, by Abcam (2 mentions)
Pro caspase 1, by Abcam (2 mentions)
Nlrp3, by Abcam (2 mentions)
Cystatin c, by Abcam (2 mentions)
β actin, by Santa Cruz Biotechnology (2 mentions)
Cleaved caspase 3, by Cell Signaling Technology (2 mentions)
Tnf α, by Abcam (2 mentions)
Gapdh, by Abcam (2 mentions)
Fibronectin, by Abcam (2 mentions)
35 protocols using kim 1
1
Immunohistochemical Analysis of Kidney Biomarkers
2
Histological Analysis of Kidney Injury
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After harvesting, kidneys were immediately fixed in 4% paraformaldehyde and then embedded in paraffin. The tissues were sectioned and stained with hematoxylin & eosin (H&E) stain, periodic acid Schiff (PAS) stain, and Masson’s trichrome stain. Images were captured using a confocal microscope (Nikon, Tokyo, Japan). Tubular injury in PAS-stained sections was assessed and scored at a ×200 magnification using 10 randomly selected fields for each kidney as follows: 0, 0%; 1, ≤10%; 2, 11–25%; 3, 26–45%; 4, 46–75%; and 5, 76–100% [18 (link)]. For IHC staining, the sections were incubated with primary antibodies against kidney injury molecule-1 (Kim-1; Abcam, Cambridge, MA, USA), neutrophil gelatinase-associated lipocalin (NGAL; Santa Cruz Biotechnology, Santa Cruz, CA, USA), 4-hydroxynonenal (4-HNE; Abcam), galectin-3 (Abcam), α-smooth muscle actin (α-SMA; Sigma-Aldrich), or collagen I (Abcam) overnight at 4 °C and then probed with a secondary antibody for 30 min at room temperature. All the sections were counterstained with hematoxylin. The percentage of positive staining was assessed in 5 randomly selected fields (×400 magnification) per each kidney using i-Solution Lite V.9.1 image analysis software (IMTechnology, Vancouver, BC, Canada). Galectin 3 positive cells or CD4 positive cells were counted in 5 randomly selected fields (×400 magnification) per each kidney.
3
Physiologic Parameters in Aging Mice
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Physiologic parameters were measured just before the mice were killed at 6 wk of age. Body weight was examined, blood pressure was measured using the tail-cuff method (BP-98A; Softron, Tokyo, Japan), and urine samples after 24-h food withdrawal were collected in metabolic cages. Blood samples were obtained using a 21-gauge needle inserted into the right atrium of mice that were unfed for 24 h. Urinary kidney injury molecule-1 (KIM-1; Abcam, Cambridge, MA, USA) and neutrophil gelatinase-associated lipocalin (NGAL; Abcam) levels were measured using an enzymatic method.
4
Histological Assessment of Kidney Injury
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Kidneys were rapidly removed from each mouse and then fixed in 4% phosphate-buffered paraformaldehyde. The tissues were embedded in paraffin and then thin sections were made from the paraffin blocks. The sections were incubated with hematoxylin and eosin (H&E) stain and periodic acid Schiff (PAS) stain. Images were captured using the NIKON A1+ confocal microscope (Nikon, Tokyo, Japan). The degree of tubular injury was scored as previously described [18 (link)]. For immunohistochemical staining, the kidney sections were probed with antibodies against kidney injury molecule-1 (Kim-1; Abcam, Cambridge, MA, USA), neutrophil gelatinase-associated lipocalin (NGAL; Santa Cruz Biotechnology), 4-hydroxynonenal (4-HNE; Abcam), or Galectin-3 (Abcam).
5
Renal Injury Protein Profiling
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Mouse renal tissues were collected and homogenized in lysis buffer. The homogenates were centrifuged at 16,000×g at 4°C for 15 min. A bicinchoninic acid protein assay kit (Pierce Co, Rockford, IL, USA) was used to analyze the protein concentrations. Equal amounts (20 µg) of the protein were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis geland transferred to a polyvinylidene difluoride membrane. The membranes were blocked with 5% nonfat dry milk in Tris-buffered saline and incubated with the following primary antibodies overnight: cleaved caspase-3 (1:1000; Abcam, Cambridge, MA, USA), KIM-1 (1:1,000; Abcam, Cambridge, MA, USA), NGAL (1:1,000; Abcam, Cambridge, MA, USA), collagen I (1:1,000; Abcam, Cambridge, MA, USA), HIF-1α (1:1,000; Novus Bio, Littleton, CO, USA), HO-1 (1:1,000; BD transduction, San Jose, CA, USA), ferritin heavy chain (1:1000; Abcam, Cambridge, MA, USA), gp91 phox (1:1,000; BD transduction, San Jose, CA, USA), GPX4 (1:1,000; Abcam, Cambridge, MA, USA), and β-actin (1:1,000; Cell Signaling, Danvers, MA, USA). After washed, the membranes were incubated for 2 h with a secondary antibody coupled to horseradish peroxidase (1:5,000; Santa Cruz, CA, USA). Densitometric analyses were carried out with image acquisition and analysis software (Bio-Rad).
6
Immunohistochemical Analysis of Apoptosis and Kidney Injury
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According to the manufacturer’s instructions (Boshide, Wuhan, China), the heart and kidney sections were first dewaxed and rehydrated. The endogenous peroxidase activity was blocked with 3% hydrogen peroxidase for 10 min. Then the sections were incubated with goat serum blocking solution at room temperature for 20 min(m). The sections were then incubated with primary antibodies for Apoptosis Stimulating Fragment (FAS, Sigma, St. Louis, MO, USA) or Kidney Injury Molecule 1 (KIM-1, ABCAM, Cambridge, MA, USA), overnight at 4 °C. After being rinsed, the tissue sections were reacted with peroxidase-conjugated goat anti-rabbit IgG secondary antibodies at 37 °C for 30 min. Finally, all sections were dehydrated, cleared, mounted, and visualized with a DAB-based colorimetric method and Image Pro Plus 6.0 analysis software (Media Cybernetics Inc., MD, USA). The IHC index was defined as the average integral optical density.
7
Immunohistochemical Analysis of Kidney Pathology
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Paraffin kidney sections from mice or humans (4 μm) were deparaffinized and rehydrated, and antigen retrieval was conduction utilizing 10 mM sodium citrate or EDTA for 15 min within a microwave oven. After blocking with 3% H2O2/PBS and 5% bovine serum albumin (BSA)/PBS, the following primary antibodies were incubated overnight at 4 °C: rabbit mouse anti-human/mouse LIGHT, LTβR, KIM-1, 3-NIT, 4-Hydro, Drp1, Mfn2, Bcl2, α-SMA (1:200, Abcam, Cambridge, UK), and rabbit anti-mouse/human HVEM (1:200, Santa Cruz, USA). Sections were then incubated with horseradish peroxidase-labeled goat anti-rabbit/mouse secondary antibodies (1:800, Beyotime, Shanghai, China) for 1 h at 25 °C. Following washing three times with PBS, 3,3’-diaminobenzidine chromogen solution (DAB, Beyotime, Shanghai, China) was used for visualization, and the sections were counterstained with hematoxylin and analyzed with light microscopy (Olympus, Tokyo, Japan). At least six viewing fields were randomly selected to quantify the number of positive areas (0.04 mm2).
8
Ferroptosis Regulation in Kidney Injury
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FG-4592 was purchased from Selleck (Houston, Texas, USA), while wortmannin and antibodies to p-Akt, Akt, p-GSK-3β, GSK-3β, IL-1β, and F4/80 were purchased from CST (Danvers, MA, USA). FA was obtained from Dalian Meilun Biotechnology Co. (Dalian, China), and Fer-1 was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies to β-actin, HIF-1α, KIM-1, Nrf2, GPX4, 4-HNE, vimentin, fibronectin (Fn), SLC7A11, ferroportin, histone H3, and assay kits for Perls' iron staining were acquired from Abcam (Cambridge, MA, USA). Antibody to MDA was purchased from Novus Biologicals (Littleton, CO, USA). Antibodies to TNF-α, HO-1, and collagen IV were obtained from Proteintech (Wuhan, China). Antibody to Keap1 was acquired from Wanlei (Shenyang, China). Assay kits for GSH (A-006-2) and MDA (BC0025) were acquired from Solarbio (Beijing, China). Assay kits for creatinine (C011-2), urea nitrogen (BUN) (C013-2), and iron (A039-2) were obtained from Jiancheng (Nanjing, China). TUNEL assay was acquired from Roche (Basel, Switzerland). An assay kit for nuclear extract was purchased from Active Motif (Tokyo, Japan).
9
Western Blot Analysis of Inflammatory Markers
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Samples were homogenized in lysis buffer, and the supernatant was extracted for measurement of total protein with a Protein Assay Kit (BCA; Pierce, Santa Cruz, CA, USA). Equal amounts of total protein were separated in SDS-PAGE and transferred to PVDF membranes in Trisglycine methanol buffer. The bands were visualized using the enhanced chemiluminescence. GADPH was developed as a loading control to normalize the data. The antibodies against NLRP3, ASC, pro-caspase-1, IL-1β, Kim-1 and IL-18 were purchased from Abcam (Cambridge, MA, USA). Antibody against BCL6 was obtained from Cell Signaling Technology (Beverly, MA, USA). Antibodies against NGAL and Cystatin C were purchased from Bioworld Technology, Ltd (Nanjing, Jiangsu, China). Antibody against GAPDH was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
10
Cisplatin-Induced Kidney Injury Model
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CDDP was purchased from Sigma-Aldrich (St. Louis, MO, USA) and was used to induce kidney injury. CDDP was dissolved in normal saline (0.9% sodium chloride). Primary antibodies against neutrophil gelatinase-associated lipocalin (NGAL), kidney injury molecule-1 (KIM-1), selenium binding protein-1 (SBP1), and Ki-67 were purchased from Abcam (Cambridge, MA, USA). Assay kits used to measure BUN and sCr were purchased from Abcam. Superoxide dismutase (SOD) and catalase assay kits were purchased from Cayman Chemical (Ann Arbor, MI, USA). TUNEL assay kits were purchased from Promega (Promega, Madison, WI, USA). All other chemicals used in this study were purchased from Sigma-Aldrich.
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