First, specimens were fixed with formalin, embedded in paraffin, sectioned (thickness, 4 mm) and transferred to microscope slides. Sections were dewaxed in xylene followed by rehydration in a graded series of ethanol solutions. Then, they were incubated in citrate buffer (pH 6.0) and heated to 121 °C in an autoclave to retrieve the antigen. Endogenous peroxidase was inactivated after immersion into 0.3% hydrogen peroxide for 30 min. To eliminate nonspecific binding, specimens were incubated in 10% goat serum for 1 h at room temperature. Subsequently, sections were incubated with primary antibody: anti-CD-31 antibody (1:100; Abcam) and anti-nephrin antibody (1:100; Abcam) overnight. Immunoreactive staining was processed using the peroxidase–anti-peroxidase method according to manufacturer instructions (DAKO, Hamburg, Germany). 3, 39-diaminobenzidine tetrachloride (DAB) chromogen solution was used to detect the reaction. After rinsing in water for 30 min, sections were counterstained with hematoxylin, and dehydrated. Finally, they were mounted in mounting medium for interpretation. For all antibodies, negative controls were used in which the primary antibody was omitted. All sections were negative.
Anti nephrin antibody
Manufactured by Abcam
Sourced in United States
Anti-nephrin antibody is a laboratory reagent used for the detection and study of the nephrin protein. Nephrin is a key component of the glomerular filtration barrier in the kidney. This antibody can be used in techniques such as Western blotting, immunohistochemistry, and immunocytochemistry to identify and analyze the nephrin protein in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti wt 1 antibody, by Abcam (2 mentions)
Anti podocin antibody, by Abcam (2 mentions)
Anti cd31 antibody, by Abcam (1 mentions)
Anti cleaved caspase 3 antibody, by Abcam (1 mentions)
Anti mettl3 antibody, by Abcam (1 mentions)
Anti phospho nf κb p65 antibody, by Cell Signaling Technology (1 mentions)
Anti mmp2 antibody, by Proteintech (1 mentions)
Anti fibronectin antibody, by Abcam (1 mentions)
Anti mettl14 antibody, by Cell Signaling Technology (1 mentions)
Anti igf2bp2 antibody, by Proteintech (1 mentions)
Anti wtap antibody, by Proteintech (1 mentions)
Anti nf κb p65 antibody, by Cell Signaling Technology (1 mentions)
Anti β actin antibody, by Proteintech (1 mentions)
Streptozotocin (stz), by Merck Group (1 mentions)
Anti m6a antibody, by Synaptic Systems (1 mentions)
Lipofectamine rnaimax reagent, by Thermo Fisher Scientific (1 mentions)
Mouse albumin elisa kit, by Abcam (1 mentions)
Anti wilms tumor protein, by Abcam (1 mentions)
Transfer apparatus, by Bio-Rad (1 mentions)
Tbs t, by Bio-Rad (1 mentions)
5 protocols using anti nephrin antibody
1
Immunohistochemical Analysis of Kidney Tissue
2
Investigating the Role of m6A Modification in Diabetic Nephropathy
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The following reagents were used: transfection was performed with Lipofectamine RNAiMAX Reagent (Invitrogen Life Technologies, 13, 778-030) according to the manufacturer’s protocol. The following antibodies were used: anti-β-actin antibody (Proteintech, 66009-1-Ig); anti-m6A antibody (Synaptic Systems, 202,003); anti-METTL3 antibody (Abcam, Ab195352, ZEN-BIOSCIENCE [382,974]); anti-METTL14 antibody (Cell Signaling Technology, 51, 104); anti-WTAP antibody (Proteintech, 10200-1-AP); anti-KIAA1429 antibody (SAB, 29, 774); anti-TIMP2 antibody (Abcam, ab180630); anti-NF-κB p65 antibody (Cell Signaling Technology, 8242); anti-phospho-NF-κB p65 antibody (Cell Signaling Technology, 3033); anti-cleaved caspase3 antibody (Abcam, Ab2302); anti-NOTCH3 antibody (Proteintech, 55114-1-AP); anti-NOTCH4 antibody (Affinity, DF13597); anti-WT-1 antibody (Abcam, ab267377); anti-nephrin antibody (Abcam, ab216341); anti-podocin antibody (Abcam, ab181143); anti-MMP2 antibody (Proteintech, 10373-2-AP); anti-MT1-MMP antibody (Proteintech, 14552-1-AP); anti-IGF2BP2 antibody (Proteintech, 11601-1-AP); anti-col-Ⅳ antibody (Abcam, ab6586); and anti-fibronectin antibody (Abcam, ab6586). STZ was purchased from Sigma Chemical Company (MO, USA). The PAS kits were obtained from Solarbio (Beijing, China). Mouse Albumin ELISA Kit was obtained from Abcam Biotechnology (Cambridge, UK).
3
Extracellular Vesicle Protein Profiling
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Extracellular vesicles were isolated by ultracentrifugation and identified by the expression of CD9, CD81, CD63 AND HSP70 markers in western blot. Extracellular vesicle extracts were fractionated by SDS-PAGE and transferred to a polyvinylidene difluoride membrane using a transfer apparatus according to the manufacturer’s protocols (Bio-Rad, USA). After incubation with 5% nonfat milk in TBST (Bio-Rad, USA) (10 mM Tris, pH 8.0, 150 mM NaCl, 0.5% Tween 20) for 60 min. The membrane was washed once with TBST and incubated with antibodies against CD9 (1:1000), CD81 (1:1000), CD63 (1:1000), HSP70 (1:1000) (Sysetem Biosciences, USA), anti-podocalyxin (PODXL, 1:1000), anti-wilms tumor protein (1:1000) and anti-Nephrin antibody (1:1000) at 4 °C for 12 h (Abcam, USA). Membranes were washed three times for 10 min and incubated with a 1:20,000 dilution of horseradish peroxidase-conjugated goat anti-rabbit antibody for 90 min at room temperature. Blots were washed with TBST three times and developed with Pierce ECL kit (ThemoFisher Scientific, USA).
4
Immunoblotting of Cell Protein Markers
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After electrophoresis of total cell proteins, samples were immunoblotted as previously reported82 (link),83 (link). Membranes were then incubated overnight at 4 °C with the following polyclonal antibodies [dilution, -fold]: anti-VCAM1 antibody (Abcam,) [5000], anti-E-Cadherin antibody (BS Transduction Laboratories) [1000], anti-tenascin-C antibody (Santa Cruz Biotechnology, Santa Cruz CA) [500], anti- NPHS2 antibody (Abcam) [5000], anti-nephrin antibody (Abcam) [5000] and anti-tubulin (Sigma–Aldrich, Saint Louis, MO [5000]. Anti-vimentin (Santa Cruz Biotechnology) [1000], anti-cofilin-1 (Santa Cruz Biotechnology) [1000], anti-vinculin (Santa Cruz Biotechnology) [1000], anti-ILK (Santa Cruz Biotechnology) [1000], anti-β-catenin (Santa Cruz Biotechnology) [2000].
Coomassie staining (Sigma) of the membrane was used as an internal loading control. Blots were analyzed by densitometric scanning with Image J. Western blot studies in cultured cells were performed in at least three independent experiments, and a representative figure is shown.
Coomassie staining (Sigma) of the membrane was used as an internal loading control. Blots were analyzed by densitometric scanning with Image J. Western blot studies in cultured cells were performed in at least three independent experiments, and a representative figure is shown.
5
Immunofluorescence Analysis of Glomerular Markers
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Immunofluorescence was performed using frozen Sections (10 µm). The following antibodies were used as primary antibodies: monoclonal rat anti-CD31 antibody (BD Biosciences); polyclonal rabbit anti-type IV collagen antibody (Abcam); anti-mouse Alk1 antibody (R&D systems); anti-Nephrin antibody (Abcam); anti-WT1 antibody (Abcam); anti-podocin antibody (Abcam); anti-cleaved-caspase 3 (Cell Signaling); anti-PDGFRB (R&D). Alexa Fluor 488 or 647 conjugated antibodies (ThermoFisher Scientific) were used as secondary reagents and slides were mounted with Fluoroshield/DAPI (Sigma). Images were obtained by confocal microscopy (Olympus Fluoview). For quantification of immunofluorescence, staining intensity and area was quantified using 50 randomly selected glomeruli per kidney section. Brightness and contrast were adjusted on displayed images (identically for compared image sets) and quantified (identical threshold settings for compared image sets) using ImageJ. For patient samples, paraffin-embedded tissues were cut into 4- to 6-μm sections and processed for immunofluorescence. Antigen retrieval was performed in citrate solution pH = 6. The sections were then labeled with anti-human Alk1 antibody (R&D systems). Slides were subsequently exposed to specific AF647-conjugated secondary antibody (ThermoFisher).
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