IF was performed according to our previously published protocols [17 (link),18 (link)]. Kidney sections were blocked with 10% horse serum for 1 h. Kidney tissue slides were probed with primary antibodies CD206 (Abcam, Cambridge, UK), HO-1(Abcam, Cambridge, UK), synaptopodin (Abcam, Cambridge, UK), or F4/80 (Santa Cruz, Dallas, TX, USA) and incubated at 4 °C overnight. Kidney tissue slides were subsequently incubated with fluorescent secondary antibodies (Invitrogen, Carlsbad, CA, USA). Ten glomeruli in each section were randomly selected for the Olympus confocal microscope (Olympus, Tokyo, Japan). Six regions within renal glomeruli from three sections obtained from six rats were detected. Percentage of positive labeled cells was calculated as percentage of the area (Image-Pro Plus software, Media Cybernetics, Silver Spring, MD, USA).
Synaptopodin
Manufactured by Abcam
Sourced in United Kingdom, United States
Synaptopodin is a protein involved in the structure and function of podocytes, specialized cells found in the kidney glomerulus. It plays a role in the maintenance of the glomerular filtration barrier. Synaptopodin is commonly used as a marker for podocyte identification and assessment of glomerular health.
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Lab products found in correlation
Gapdh, by Cell Signaling Technology (2 mentions)
Bcl 2, by Abcam (2 mentions)
Bcl2 associated x (bax), by Abcam (2 mentions)
Podocin, by Abcam (2 mentions)
Fluorescent secondary antibody, by Thermo Fisher Scientific (1 mentions)
F4 80, by Santa Cruz Biotechnology (1 mentions)
Cd206, by Abcam (1 mentions)
Confocal microscope, by Olympus (1 mentions)
Image pro plus software, by Media Cybernetics (1 mentions)
Alexa fluor 488, by Abcam (1 mentions)
Eclipse e200, by Nikon (1 mentions)
Ecl western blotting detection system, by Cytiva (1 mentions)
Jagged 1, by Cell Signaling Technology (1 mentions)
Cleaved notch1, by Abcam (1 mentions)
Bca protein assay, by Beyotime (1 mentions)
Vascular endothelial growth factor (vegf), by Abcam (1 mentions)
Mmp9 elisa kits, by Abcam (1 mentions)
Mmp 9, by Abcam (1 mentions)
Sb203580, by Selleck Chemicals (1 mentions)
Dylight 594 conjugated secondary antibodies, by Thermo Fisher Scientific (1 mentions)
10 protocols using synaptopodin
1
Immunofluorescence Analysis of Kidney Tissue
2
Podocyturia Quantification in Fabry Disease
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We have previously described the method to study podocyturia in detail in patients with Fabry disease [15 ]. Briefly, a midstream freshly voided urine sample was collected on-site after a minimum of 3 h without voiding; 20 mL of urine were centrifuged at 700 g for 5 min using a cytospin. The supernatant was discarded, and the obtained sediment was stored in 100-µL aliquots at room temperature and mixed with a 1.5-mL solution made of 40% paraformaldehyde diluted in phosphate-buffered saline (PBS; pH 7.2–7.4) to reach a final 10% concentration. Nuclei of podocytes were stained with 4′,6-diamidino-2-phenylindole (DAPI). Podocytes were identified by immunofluorescence using synaptopodin as the primary antibody (1: 100, Abcam, Cambridge, MA, USA) and IgG anti-rabbit Alexa Fluor® 488 (1: 100, Abcam) as the secondary antibody. Samples were analyzed employing an epifluorescent microscope Nikon Eclipse E 200. Following our standardized technique, podocytes were counted in 10 randomly chosen 20× fields of the slides, and the average of the counted podocytes in the microscopy fields was considered as the final count for each subject. The results were corrected based on the levels of urinary creatinine found in each sample [15 ].
3
Protein Expression Analysis of Notch Signaling
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The concentration of extracted protein was detected by Protein BCA Assay (Beyotime, Jiangsu, China). Protein was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes. After blocking with nonfat milk, primary antibodies specific for Jagged-1 (Cell Signaling Technology, CST, Boston, USA), full-length Notch-1 (CST), cleaved Notch-1 (NICD, Abcam, Cambridge, UK), Synaptopodin (Abcam), Hes-1 (Abcam), VEGF (Abcam), p53 (CST), p62 (Abcam), LC3B (Abcam) were used. Goat anti-rabbit IgG conjugated with horseradish peroxidase was used to detect the expression of primary antibodies, and protein bands were visualized by ECL western blotting detection system (Amersham, Little Chalfont, UK).
4
Antibody Validation for Podocyte Proteins
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Antibodies specific for podocin, synaptopodin, vimentin, mmp9, WT1 and phospho‐FAK (phospho Y397) were from Abcam (Cambridge, MA, USA). Anti‐NEPH1 antibodies purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Anti‐p38 MAPK, phospho‐p38 MAPK (Thr180/Tyr182) and FAK antibodies were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Anti‐β‐actin antibodies were obtained from Proteintech (Chicago, IL, USA). Secondary antibodies were from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA, USA) and Dylight 594 conjugated secondary antibodies were from Pierce, Thermo Fisher Scientific Inc. (Rockford, IL, USA). mmp9 ELISA kits were from Abcam. The p38 inhibitor SB203580 was from Selleck (Houston, TX, USA). LDL and FITC‐Phalloidin were purchased from Sigma Chemical Company (St Louis, MO, USA). The FAK inhibitor TAE226 was from Novartis Pharmaceuticals (Plantation, FL, USA). For in vitro use, SB203580 was first diluted in DMSO as a stock liquid (50 mM); for further use, the stock liquid was added for a terminal concentration at 5 μg/ml 26 . For oxidation, LDL (5 mg/ml) were mixed with 5 μmol CuSO4, incubated for 18 hrs at 37°C, and oxidation was evaluated as described previously 27 .
5
Angiotensin II-Induced Kidney Damage Protocol
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Mini-osmotic pumps were purchased from DURECT Corporation (Model 2006, Cupertino, CA). Angiotensin II (AngII), Honokiol (HKL), FITC-inulin, Bovine Serum Albumin (BSA) were purchased from Sigma Aldrich (Sigma Aldrich, USA). Mouse urine creatinine assay kit was purchased from R&D Systems (R&D, USA). Mouse urine albumin, BUN and Scr assay kit were purchased from AssayPro Corporation (AssayPro, USA). Antibodies against SIRT3 (28kDa), acetylated lysine, Tubulin and GAPDH were purchased from Cell Signaling Technology (CST, USA). Antibodies against KLF15, fibronectin, collagen type IV, synaptopodin, WT-1 were purchased from abcam (Abcam, USA).
6
Protein Analysis of Mouse Renal Cortex and Podocytes
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Protein analysis was performed on mouse renal cortex tissues (Ji et al., 2017 (link)) and cultured podocytes as described previously. EGFR, p-EGFR and cleaved caspase three antibodies were purchased from Cell Signaling (Beverly, MA, United States). ERK1/2, p-ERK1/2, Bcl2, Bax and synaptopodin antibodies were purchased from Abcam (Cambridge, United Kingdom). β-actin antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, United States).
7
Protein Expression Analysis in Podocytes
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MCP5 podocytes were lysed in RIPA lysis buffer (Beyotime), supplemented with protease inhibitor mixture (Beyotime). The protein samples were separated by SDS-PAGE, followed by transferring to PVDF membranes (Millipore, USA) and blocked with 5% non-fat milk at room temperature for 2 h. The membranes were incubated overnight with primary antibodies at 4°C then incubated with secondary antibodies at room temperature for 2 h. The primary antibodies used for Western blotting were as follows: KLF9 (Abcam, 1:1000), Bcl-2 (CST, 1:1000), Bax (CST, 1:1000), (cleaved) caspase 3 (Abcam, 1:1000), synaptopodin (Abcam, 1:1000), podocin (Abcam, 1:1000) and GAPDH (Abcam, 1:1000). After washing with TBST, the membrane was incubated with appropriate secondary antibodies (Abcam, 1:10,000). Blots were visualized by the enhanced chemiluminescence system (Santa Cruz Biotechnology Inc., CA, USA).
8
Renal Cortex Protein Profiling
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The proteins of the renal cortex were lysed with lysis buffer containing protease inhibitors and quantified with a BCA protein assay kit (BN27109-500T, Biorigin, Beijing, China). Total protein samples were separated on 10% polyacrylamide sodium dodecyl sulfate gels and then transferred to nitrocellulose membranes. Afterwards, the membranes were blocked with 5% skimmed milk powder dissolved in Tris-Buffered Saline and Tween 20 (TBST) for 1 h, followed by incubation with the primary antibodies synaptopodin (ab32127, abcam, Cambridge, UK), AKT (#4685, CST, Boston, USA), p-AKT (#13038, CST, Boston, USA), PI3K (#4257, CST, Boston, USA), p-PI3K (#17366, CST, Boston, USA), mTOR (#2983, CST, Boston, USA) and p-mTOR (#5536, CST, Boston, USA) overnight at 4 °C. After washing with TBST, the membranes were incubated with HRP-conjugated secondary antibodies (donkey anti-mouse, goat anti-rabbit) for 1 h. Finally, an ECL Kit (P1010, Applygen, Beijing, China) was used to detect positive binding under a multi-functional imaging system (Tanon 5200, Shanghai, China). All protein bands were quantified using Image J software.
9
Antibody Characterization for Podocyte Proteins
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Monoclonal anti-ezrin antibody was purchased from LifeSpan Bioscience Inc. (Seatle, WA). Polyclonal anti-ezrin, ERM, phospho-ERM, β-actin and GAPDH antibodies were purchased from Cell Signaling (Danvers, MA). Goat anti-mouse podocalyxin antibody was purchased from R&D Systems (Minneapolis, MN). Polyclonal anti-Rho-GDIα antibodies were purchased from Sigma Aldrich (St. Louis, MO). Polyclonal anti-radixin, moesin, podocin and synaptopodin antibodies were purchased from Abcam (Cambridge, MA). Polyclonal anti-NHERF2 antibody was purchased from Thermo Fischer Scientific (Waltham, MA). Polyclonal anti-CLIC5 antibody was purchased from Alomone Labs (Jerusalem, Israel). For immunoblotting, all antibodies were diluted with Solution 1 (Can Get Signal; TOYOBO, Osaka, Japan) by a factor of 1:1000. For immunofluorescence analysis, all antibodies were diluted with Solution A (Can Get Signal; TOYOBO, Osaka, Japan) by a factor of 1:100.
10
Characterization of Recellularized Kidney Scaffolds
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All samples were fixed in 10% v/v buffered neutral formalin, and samples were stained with haematoxylin and eosin (H+E) for light microscopy.
IHC was performed to characterise cell types and their distribution on the recellularised kidney bioscaffolds; the protocol is the same as above. Primary antibody dilutions were: aquaporin-1, aquaporin-2, synaptopodin, and von Willebrand Factor (all Abcam, UK) -all at 1:500. For all experimental work as described above, n ≥ 3.
IHC was performed to characterise cell types and their distribution on the recellularised kidney bioscaffolds; the protocol is the same as above. Primary antibody dilutions were: aquaporin-1, aquaporin-2, synaptopodin, and von Willebrand Factor (all Abcam, UK) -all at 1:500. For all experimental work as described above, n ≥ 3.
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