Nephrin

Manufactured by Thermo Fisher Scientific

Sourced in United States

Nephrin is a laboratory equipment product used in research applications. It is a protein that plays a critical role in the function of the kidney's filtration system. The core function of Nephrin is to maintain the structural integrity and permeability of the glomerular filtration barrier in the kidney.

Automatically generated - may contain errors

Lab products found in correlation

9 protocols using nephrin

Immunofluorescence Analysis of Kidney Injury

Check if the same lab product or an alternative is used in the 5 most similar protocols

TUNEL kits (Servicebio, G1501) were used to detect apoptotic cells in paraformaldehyde-fixed kidney tissues, and the appropriate experimental protocols were applied. For immunofluorescence in tissues, paraffin sections were blocked with 10% donkey serum and incubated overnight at 4 °C with primary antibodies nephrin (1:100, Invitrogen, PA5-106921), podocin(1:100, Proteintech, 20384-1-AP), cd2ap(1:100, Invitrogen, PA5-51894), TNF-α(1:500, Servicebio, GB11188), IL-6(1:500, Servicebio, GB11117), IL-1β(1:300, Servicebio, GB11113), and LC3(1:500, Proteintech, 14600-1-AP), followed by secondary antibodies. Finally, the nuclei were restained with DAPI and observed under a confocal microscope (Olympus, FV3000).
For immunofluorescence in cells, each group of cells were given the corresponding treatment and sequentially subjected to cell rupture, serum closure, incubated with the corresponding primary antibodies nephrin (1:100, Invitrogen, PA5-106921), podocin (1:100, Proteintech, 20384-1-AP), cd2ap (1:100, Invitrogen, PA5-51894), and LC3(1:500, Proteintech, 14600-1-AP) overnight at 4 °C and secondary antibodies. Finally, after DAPI restaining nuclei, the cells were observed under confocal microscopy.

Ji B., Liu J., Yin Y., Xu H., Shen Q, & Yu J. (2023). Minnelide combined with anti-ANGPTL3-FLD monoclonal antibody completely protects mice with adriamycin nephropathy by promoting autophagy and inhibiting apoptosis. Cell Death & Disease, 14(9), 601.

+ Open protocol

+ Expand

Detailed Methodology for Studying Podocyte Apoptosis

Check if the same lab product or an alternative is used in the 5 most similar protocols

Butaprost and AH6809 were purchased from Caymen (USA), Recombinant human TGF-β1 was purchased from PeproTech (UK), The CCK-8 kit and Trizol RNA extracting kit were purchased from Invitrogen (USA). Mouse cAMP and PGE2 ELISA kit were purchased from Weston Biology Company (Shanghai, China). The reverse transcription (RT) kit was purchased from Fermentas (USA). The real-time quantitative PCR (RT-qPCR) kit was from Roche (USA). The Annexin V-FITC cell apoptosis assay kit was purchased from Beyotime (Shanghai, China). Nephrin, podocin, and CD2AP primers were purchased from Invitrogen (USA). Rabbit anti-mouse polyclonal antibodies for Nephrin, podocin and caspase3 were purchased from Abcam (UK). Rabbit anti-rat monoclonal antibodies of GAPDH, CD2AP, PI3K-p85 and Akt were purchased from Cell Signaling (USA), as well as phosphorylated PI3K-p85 and Akt monoclonal antibodies. The horseradish peroxidase (HRP) labeled goat anti-mouse and goat anti-rabbit IgG secondary antibody were purchased from SantaCruz (USA). Alexa Fluor 488 labeled goat anti-rabbit IgG (H+L) was purchased from Fcmacs (Nanjing, Jiangsu province, China).

Liu J., Zhang Y.D., Chen X.L., Zhu X.L., Chen X., Wu J.H, & Guo N.F. (2018). The protective effect of the EP2 receptor on TGF-β1 induced podocyte injury via the PI3K / Akt signaling pathway. PLoS ONE, 13(5), e0197158.

+ Open protocol

+ Expand

Kidney Podocyte Protein Expression Analysis

Check if the same lab product or an alternative is used in the 5 most similar protocols

Kidney tissues and podocytes were first lysed with RIPA, followed by electrophoresis, membrane transfer, blocking, probing with primary antibodies nephrin (1:500, Invitrogen, PA5-106921), podocin (1:500, Proteintech, 20384-1-AP), cd2ap (1:500, Invitrogen, PA5-51894), Bax (1:500, Abcam, ab216494), Bcl-2 (1:500, Abcam, ab196495), Cleaved-caspase3 (1:500, Servicebio, GB11532), p-mTOR (1:1000,CST,5536 T), mTOR (1:1000,CST,2983 T), Beclin1(1:500, Servicebio, GB112053), p62(1:500, Servicebio, GB11239-1), and LC3(1:1000, Proteintech, 14600-1-AP) overnight at 4 °C, and incubation with secondary antibodies for 1 h at room temperature. Finally, the immunoreactive bands were developed by chemiluminescence and analyzed using Image J software.

+ Open protocol

+ Expand

Immunofluorescence Analysis of Renal Pathology

Check if the same lab product or an alternative is used in the 5 most similar protocols

Frozen renal specimens underwent an initial blocking process. Subsequently, sections were subjected to overnight incubation with primary antibodies, including IgG-FITC (Abcam, MA, USA), C3 (MP Biomedicals, CA, USA), TGF-β1 (CellSignalingTechnology, MA, USA), Smad3 (CellSignalingTechnology), nephrin (Invitrogen, CA, USA), SYNPO (Invitrogen), CD19 (CellSignalingTechnology), CD138 (CellSignalingTechnology), SIRT1 (CellSignalingTechnology), and AMPK (CellSignalingTechnology). After primary antibody incubation, the sections were treated with secondary antibodies, Alexa Fluor anti-rabbit 488 or Alexa Fluor anti-mouse 594 (Invitrogen), for 1 h. Nuclear staining was achieved using DAPI (Sigma-Aldrich, MO, USA). Assessment of MPC-5 cell apoptosis was conducted using the Click-It TUNEL kit (Invitrogen), following instructions. Finally, the specimens were visualized using an LSM700 Carl Zeiss confocal microscope (Jena, Germany).

Song Z., Jin M., Wang S., Wu Y., Huang Q., Xu W., Fan Y, & Tian F. (2023). Reciprocal regulation of SIRT1 and AMPK by Ginsenoside compound K impedes the conversion from plasma cells to mitigate for podocyte injury in MRL/lpr mice in a B cell-specific manner. Journal of Ginseng Research, 48(2), 190-201.

+ Open protocol

+ Expand

Western Blot Analysis of Renal and Cell Samples

Check if the same lab product or an alternative is used in the 5 most similar protocols

Renal samples and Namalwa cells were homogenized and lysed in RIPA buffer. Denatured proteins from the lysates were separated on SDS-PAGE gels and then transferred onto PVDF membranes. After blocking, the membranes were incubated overnight at 4 °C with primary antibodies: nephrin (Invitrogen), SYNPO (Invitrogen), SIRT1 (CellSignalingTechnology), AMPK (CellSignalingTechnology), pAMPK (CellSignalingTechnology), CPT1A (CellSignalingTechnology), and GAPDH (ThermoFisherScientific, MA, USA). Following primary antibody incubation, secondary antibodies were applied.

+ Open protocol

+ Expand

Quantifying Podocyte Gene Expression

Check if the same lab product or an alternative is used in the 5 most similar protocols

The extraction of total RNA from MPC5 podocytes was performed using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA), after which the RNA quality was detected using the NanoDrop 2000c spectrophotometer (Thermo Scientific, Waltham, USA), in adherence with the manufacturer’s protocols. Next, the RNA was converted into complementary DNA (cDNA) using the RevertAid First Strand cDNA Synthesis kit (Thermo), and qRT-PCR was performed on an ABI 7900 system using SYBR Green Real-Time PCR master mix (Thermo).
The primer sequences used were: GAPDH, F: 5'-T GTTCGTCATGGGTGTGAAC-3', R: 5'-ATGGCATGGACTGTGGTCAT-3'; synaptopodin, F:5'-GCTCGAATTCCGATGCAAATAAAC-3', R:5'-CAGGCCACAGTGAGATGTGAAGA-3'; nephrin, F:5'-CAGGGAAGACAGCAACAAACAA-3', R: 5'-CAGGTTTTCAGATAGAGCCCAGA-3'. All primers were synthesized by RiboBio (Guangzhou, China).

Zhao S., Feng J., Li J., Cao R., Zhang Y., Yang S, & Yin L. (2021). The RNA binding protein hnRNPK protects against adriamycin-induced podocyte injury. Annals of Translational Medicine, 9(16), 1303.

+ Open protocol

+ Expand

Immunofluorescence Staining of hiPSC and RPC

Check if the same lab product or an alternative is used in the 5 most similar protocols

For immunofluorescence staining, hiPSC colonies and RPCs were fixed and permeabilized using 4% paraformaldehyde and 1% Triton X-100. Samples were blocked with 5% bovine serum albumin (BSA) for 1 h at room temperature. hiPSC colonies were stained overnight with unconjugated anti-OCT4A Rabbit mAb (Cell Signaling Technology, 1:200), anti-Nanog (Cell Signaling Technology, 1:200), anti-PAX2 (Abcam, 1:100), WT-1 (Abcam, 1:100), anti-Nephrin (Thermo Scientific, 10 μg/mL), and anti-Sinaptopodin (Thermo Scientific, 20 μg/mL). The RPCs were stained overnight with primary antibodies to PAX2 (Abcam, 1:100), WT-1 (Abcam, 1:100), Nephrin (Thermo Scientific, 10 μg/mL), and Sinaptopodin (Thermo Scientific, 20 μg/mL). On the next day, samples were stained with the Alexa Fluor 488 secondary antibody (Life Technologies, Carlsbad, CA, USA, 1:1000). Subsequently, slides were mounted with Vecta Shield Antifade Mounting Medium (Vector Laboratories, Burlingame, CA, USA) containing DAPI, and images were acquired using the EVOS FL Imaging System (Thermo Fisher Scientific, Waltham, MA, USA).

Ribeiro P.D., Lojudice F.H., Fernandes-Charpiot I.M., Baptista M.A., de Almeida Araújo S., Mendes G.E., Sogayar M.C., Abbud-Filho M, & Caldas H.C. (2020). Therapeutic potential of human induced pluripotent stem cells and renal progenitor cells in experimental chronic kidney disease. Stem Cell Research & Therapy, 11, 530.

+ Open protocol

+ Expand

Immunofluorescence Staining of hiPSCs and RPCs

Check if the same lab product or an alternative is used in the 5 most similar protocols

For immuno uorescence staining, hiPSCs colonies and RPCs were xed and permeabilized using 4% paraformaldehyde and 1% Triton X-100. Samples were blocked with 5% bovine serum albumin (BSA) for 1h at room temperature. hiPSCs colonies were stained overnight with unconjugated anti-OCT4A Rabbit mAb (Cell Signaling Technology, 1:200), anti-Nanog (Cell Signaling Technology, 1:200), anti-PAX2 (Abcam, 1:100), WT-1 (Abcam, 1:100), anti-Nephrin (Thermo Scienti c, 10 μg/mL), and anti-Sinaptopodin (Thermo Scienti c, 20 μg/mL). The RPCs were stained overnight with primary antibodies to PAX2 (Abcam, 1:100), WT-1 (Abcam, 1:100), Nephrin (Thermo Scienti c, 10 μg/mL), and Sinaptopodin (Thermo Scienti c, 20 μg/mL). On the next day, samples were stained with the Alexa Fluor 488 secondary antibody (Life Technologies, Carlsbad, CA, USA, 1:1000). Subsequently, slides were mounted with Vecta Shield Antifade Mounting Medium (Vector Laboratories, Burlingame, CA, USA) containing DAPI, and images were acquired using the EVOS FL Imaging System (Thermo Fisher Scienti c, Waltham, MA, USA).

Ribeiro P.d.C., Lojudice F.H., Fernandes-Charpiot I.M.M., Baptista M.A.S.F., Araújo S.d.A., Mendes G.E.F., Sogayar M.C., Abbud‐Filho M., & Caldas H.C. (2020). Therapeutic potential of human induced pluripotent stem cells and renal progenitor cells in experimental chronic kidney disease .

+ Open protocol

+ Expand

Immunofluorescence Staining of hiPSCs and RPCs

Check if the same lab product or an alternative is used in the 5 most similar protocols

Ribeiro P.d.C., Lojudice F.H., Fernandes-Charpiot I.M.M., Baptista M.A.S.F., Araújo S.d.A., Mendes G.E.F., Sogayar M.C., Abbud‐Filho M., & Caldas H.C. (2020). Therapeutic Potential of Human Induced Pluripotent Stem Cells and Renal Progenitor Cells in Experimental Chronic Kidney Disease.

+ Open protocol

+ Expand

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!