At the experimental end-point animals were euthanized and tissues were fresh frozen in liquid nitrogen or fixed in 4% paraformaldehyde in PBS overnight. To review histology, slides were stained by haematoxylin and eosin (H&E). Immunohistochemistry (IHC) and immunofluorescence (IF) were performed using standard protocols. Specificity of immunostaining was assessed by incubation in the absence of primary antibody. We used the following primary antibodies: RFP for Strawberry detection (ab34771, 1:400; Abcam), Aquaporin 1 (NB-600–749, 1:500; Novus Biologicals), THP (AF5175, 1:100; R&D), Aquaporin 2 (ab105171, 1:1000; Abcam), Nephrin (AF3159, 1:100; R&D), CD34 (ab8158, 1:50; Abcam), CD73 (AF4488, 1:100; R&D), GFP (ab290, 1:1000; Abcam), HIF2a (NB100-132, 1:150; Novus Biologicals), CA9 (sc-25600, 1:200; Santa Cruz), turbo-RFP (AB234, 1:500; Evrogen) and pS6 (2211, 1:200; Cell Signalling). Secondary antibodies used were conjugated to HRP (IHC) or Alexa-fluor® fluorochromes (IF). Fluorescent images were obtained by confocal laser-scanning microscopy (Leica TCS SP5).
Af3159
Manufactured by R&D Systems
Sourced in United States
AF3159 is a laboratory equipment product offered by R&D Systems. It is designed for a specific function, but a detailed description while maintaining an unbiased and factual approach cannot be provided. Additional information is not available.
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Lab products found in correlation
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Ab34771, by Abcam (1 mentions)
Ab8158, by Abcam (1 mentions)
Ab290, by Abcam (1 mentions)
Nb100 132, by Novus Biologicals (1 mentions)
Nb 600 749, by Novus Biologicals (1 mentions)
Af4488, by R&D Systems (1 mentions)
Sc 25600, by Santa Cruz Biotechnology (1 mentions)
Ab234, by Evrogen (1 mentions)
Tcs sp5, by Leica (1 mentions)
Ab6885, by Abcam (1 mentions)
Na9340, by Cytiva (1 mentions)
G9545, by Merck Group (1 mentions)
Chemidoc mp imaging system, by Bio-Rad (1 mentions)
Clarity western ecl substrate, by Bio-Rad (1 mentions)
Ab207170, by Abcam (1 mentions)
647 secondary antibodies, by Thermo Fisher Scientific (1 mentions)
Ab28364, by Abcam (1 mentions)
Ltl fluorescein, by Vector Laboratories (1 mentions)
Ix73 fluorescence microscope, by Olympus (1 mentions)
11 protocols using af3159
1
Histological and Molecular Characterization
2
Western Blot Analysis of Nephrin
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Total kidney protein lysates from controls and NPHS2‐Cre+/− were prepared with RIPA extraction buffer containing phosphatase inhibitors and protease inhibitors (Santa Cruz). Total protein concentration was measured using the BCA protein assay kit (23,225/23227, Thermo Fisher Scientific). Fifty microgram of proteins was loaded onto polyacrylamide electrophoresis gels for separation and transferred onto nitrocellulose membranes. The membranes were blocked with TBS 1X containing 0.1% of Tween (TBST) and 5% of non‐fat milk and then probed with the following antibodies: goat anti‐Nephrin (1:1000, AF3159, R&D) and rabbit anti‐GAPDH (1:20,000, G9545, Sigma). After three 10‐min washing in TBST, membranes were then incubated with horseradish peroxidase‐conjugated secondary antibodies [donkey anti‐rabbit 1/5000 (NA9340, Amersham); donkey anti‐Goat 1/5000 (ab6885, Abcam)]. The resulting bands were visualized by enhanced chemiluminescence (Clarity Western ECL substrate; Bio‐Rad, 170–5061), using a ChemiDoc MP imaging system (12,003,154, Bio‐Rad). Densitometric analysis was used for quantification with the ImageJ software.
3
Immunohistochemical Staining of Kidney Cells
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The immunohistochemical procedure was provided in the recent reports.[37 , 38 , 39 , 40 , 41 ] Briefly, monolayer cells and kidney organoids in cell culture flasks were fixed in 4% paraformaldehyde for 20 min at 4 °C, followed by 3 PBS washes. Samples were blocked with 5% goat serum in 0.1% Triton X‐100/PBS for 2–3 h at room temperature and subsequently incubated with primary antibodies overnight at 4 °C. The following antibodies and dilutions were used: goat anti‐nephrin (1:200, R&D AF3159), rabbit anti‐NCC (1:100, StressMarq SPC‐402), rabbit anti‐CD31 (1:200, Abcam ab28364), rabbit anti‐podocin (1:200, Abcam ab50339), rabbit anti‐Six2 (1:200, Proteintech #11562‐1‐AP), goat anti‐GATA‐3 (1:200, R&D AF2605‐SP), mouse anti‐E‐cadherin (1:200, Invitrogen #13‐1900), rabbit anti‐cleaved‐Notch1 (1:100, Cell Signaling #4147S), rabbit anti‐UMOD (1:100, Abcam ab207170), and LTL‐fluorescein (1:1000, Vector Laboratories FL‐1321). After 3 10‐min washes with 0.1% Triton X‐100/PBS, samples were incubated with secondary antibodies for 1 h at room temperature. Corresponding Alexa Fluor 448, 555, or 647 secondary antibodies (Invitrogen) were used at 1:1000. Images were captured using an Olympus IX73 fluorescence microscope (Japan).
4
Immunostaining of Kidney Tissues and Cells
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Frozen kidney tissues and cells were transferred to 4% paraformaldehyde and fixed at 4 °C overnight. Cells and 4-μm tissue sections were permeabilized with 0.3% Triton X-100 for 10 min and blocked with 5% donkey serum for 1 h. They were then incubated with the following primary antibodies: anti-MAD2B (1:200, ab180579, Abcam, Cambridge, MA, USA), Numb (1:50, sc-136554, Santa Cruz, CA, USA), anti-Synaptopodin (1:200, #163004, Synaptic Systems, Gottingen, Germany), and anti-Nephrin (1:100, AF3159, R&D, Minneapolis, MN, USA) overnight at 4 °C. Alexa Fluor 488 IgG and Alexa Fluor 594 IgG (1:200, Invitrogen, Carlsbad, CA, USA) were used as secondary antibodies. Nucleus was counterstained with Hoechst (Beyotime Biotechnology, Shanghai, China). Sections were observed under fluorescence microscope and images were captured at identical microscopic settings.
5
Immunofluorescence Analysis of Mouse Kidney Tissues
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Mouse kidney tissues were cut into 4-μm-thick slices using a Leica frozen slicer. The kidney slices were washed twice with phosphate-buffered saline (PBS) for 5 minutes at room temperature, blocked with 3% BSA at room temperature, and then incubated overnight at 4 °C with the primary antibodies for nephrin (0.4 μg/mL; #AF3159, RD), ZO-1 (7 μg/mL; #21773-1-AP, Proteintech), WT1 (4.8 μg/mL; #ab89901, Abcam), and desmin (1.2 μg/mL; 16520-1-AP, Proteintech). On the second day, the kidney slices were washed 3 times with PBS, for 5 minutes each time, and then incubated with Alexa Fluor 594-conjugated donkey anti-rabbit (2 μg/mL; A21207, Life Technology) or donkey anti-goat secondary Abs (2 μg/mL; A11058, Life Technology) for 1 hour at room temperature. Finally, images were taken under a Nikon A1 confocal laser microscope.
6
Nephrin Immunolabeling for Super-Resolution Imaging
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Afterwards, unspecific binding sites were blocked with blocking solution (10% donkey serum, 0.9% BSA, 0.05% Tween-20 in PBS) for 1–3 h and samples were labelled with polyclonal goat anti-nephrin antibodies (AF3159, R&D Systems) at 4 °C overnight. The next step was technique-dependent: for SMLM, anti-nephrin antibodies were diluted 1:500 in blocking solution to ensure sparsely blinking of fluorophores. For SIM and STED, primary antibodies were diluted 1:50 and for ExM 1:20 in blocking solution to guarantee dense enough labelling after expansion. Samples were washed with 1% BSA in PBS before detecting primary antibodies with Alexa Fluor 488-conjugated donkey anti-goat IgG (A-11055, Thermo Fisher Scientific) or Atto647N-conjugated rabbit anti-goat IgG (605456013, Rockland) at RT for 90 min. Secondary antibodies were diluted 1:2000 for dSTORM, 1:600 for SIM and STED, and 1:100 for ExM in blocking solution. After washing sections extensively, for ExM, nuclei were stained with Hoechst (1:600, 33342, Thermo Fisher, Scientific) for 20 min at RT and washed with PBS. Antibodies were postfixed with 2% PFA in PBS for 10 min before washing the tissue with PBS. Samples were handled under the absence of light after adding secondary antibodies.
7
Whole Mount Immunofluorescence Staining of E18.5 Kidneys
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For whole mount immunofluorescence staining of E18.5 kidneys, the iDisco staining protocol (12 (link)) (https://idisco.info/idisco-protocol/ ) with methanol pretreatment was applied using an antinephrin antibody (R&D AF3159). An incubation time n = 1 day and a solution volume of 1.6 ml were used for the relevant steps. The kidneys were mounted in 8-well glass chamber slides (Thermo Fisher, 154534) and imaged immediately using an IMIC digital microscope (FEI, Type 4001) with a Polychrome V light source, an Orca-R2 camera controller from Hamamatsu (C10600), and Live Acquisition software (FEI, version 2.6.0.14). Hundred-micrometer Z-stack images of stained whole-mount E18.5 kidneys were analyzed with the open-source image processing software Fiji (ImageJ, version 2.0.0-rc69/1.52i, https://imagej.net/Fiji ). In the TrackMate v3.8.0 plugin, the Downsample LoG detector was set to 80.0 pixel for the estimated blob diameter with a 16-pixel threshold and downsampling factor 2. The number of spots per frame was added to calculate the number of glomeruli per kidney. A 100 μm distance between frames was chosen to avoid double counting of identical glomeruli in consecutive images, given an average glomerular diameter of 80 μm.
8
Quantifying Glomeruli in E18.5 Murine Kidneys
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One-hundred micrometers Z-stack images of whole-mount E18.5 kidneys stained for nephrin (R&D AF3159) were analyzed with the open source image processing software Fiji (ImageJ, version 2.0.0-rc69/1.52i, https://imagej.net/Fiji ). In the TrackMate v3.8.0 plugin, the Downsample LoG detector was set to 80.0 pixel for the estimated blob diameter with a 16-pixel threshold and downsampling factor 2. The number of spots per frame were added to calculate the number of glomeruli per kidney. A 100 µm distance between frames was chosen to avoid double counting of identical glomeruli in consecutive images, given an average glomerular diameter of 80 µm.
9
Immunodetection of Renal Fibrosis Markers
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Rat kidney tissues were fixed in formalin, then paraffin embedded and 5 μm sections were mounted on silanized slides. Immunodetections were performed using primary anti-α-SMA (SC-130617, Santa Cruz Biotechnology) and anti-nephrin (AF3159, R&D System) in blocking solution overnight at 4°C. Immunosignals were developed using the LSAB + System − HRP (DakoCytomation). Images were analyzed by quantifying glomeruli in renal cortex using the software ImageJ with Color-Deconvolution plugin.
10
Immunostaining in 24-well Dishes
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The whole process of immunostaining was performed using a 24-well dish. Before starting the immunostaining, to prevent contamination, the samples were washed gently and thoroughly with the antibody dilution solution that was stipulated by each protocol (41–44 (link), 50 (link), 51 (link)). The samples were immersed in the primary antibody solution (750–1,000 μL) on a horizontal shaker at room temperature for two overnights. Then, the samples were washed with the antibody dilution solution of each tissue-clearing protocol for 30 min × 3. The secondary antibody solution (750–1,000 μL) was added to the sample on a horizontal shaker at room temperature for two overnights. The order of tissue clearing and IF followed each protocol’s recommendations.
The primary antibodies used in this study were goat antimice nephrin antibody (AF3159; R & D Systems, Minneapolis, MN, USA), sheep antihuman nephrin antibody (AF4269; R & D Systems), and rabbit anti-claudin-1 antibody (ab15098; Abcam, Cambridge, MA, USA). The secondary antibodies were Alexa Fluor-488 antigoat IgG antibody (Thermo Fisher Scientific, Waltham, MA, USA) and Alexa Fluor-555 antirabbit IgG antibody (Thermo Fisher Scientific). The dilution ratios of the primary and secondary antibodies were 1:100, and 1:300, respectively. DAPI (Roche, Basel, Switzerland) was used for nuclei staining.
The primary antibodies used in this study were goat antimice nephrin antibody (AF3159; R & D Systems, Minneapolis, MN, USA), sheep antihuman nephrin antibody (AF4269; R & D Systems), and rabbit anti-claudin-1 antibody (ab15098; Abcam, Cambridge, MA, USA). The secondary antibodies were Alexa Fluor-488 antigoat IgG antibody (Thermo Fisher Scientific, Waltham, MA, USA) and Alexa Fluor-555 antirabbit IgG antibody (Thermo Fisher Scientific). The dilution ratios of the primary and secondary antibodies were 1:100, and 1:300, respectively. DAPI (Roche, Basel, Switzerland) was used for nuclei staining.
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