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).
647 secondary antibodies
Manufactured by Thermo Fisher Scientific
Sourced in United States
The 647 secondary antibodies from Thermo Fisher Scientific are fluorescently-labeled antibodies designed to detect and bind to primary antibodies. They can be used in a variety of immunoassay and imaging applications to visualize target proteins or other biomolecules.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Ix73 fluorescence microscope, by Olympus (1 mentions)
Ab207170, by Abcam (1 mentions)
Af3159, by R&D Systems (1 mentions)
Ab28364, by Abcam (1 mentions)
Ltl fluorescein, by Vector Laboratories (1 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (1 mentions)
A1 standard sensitivity confocal microscope, by Nikon (1 mentions)
Alexa 488, by Thermo Fisher Scientific (1 mentions)
Dibutylphthalate polystyrene xylene (dpx), by Merck Group (1 mentions)
Texas red x phalloidin, by Thermo Fisher Scientific (1 mentions)
Rabbit anti β gal, by MP Biomedicals (1 mentions)
Vectashield, by Vector Laboratories (1 mentions)
Rabbit anti p jnk, by Cell Signaling Technology (1 mentions)
Rabbit or chicken anti gfp, by Thermo Fisher Scientific (1 mentions)
Mouse anti gluriia, by Developmental Studies Hybridoma Bank (1 mentions)
Rat anti elav, by Developmental Studies Hybridoma Bank (1 mentions)
Anti ha monoclonal antibody, by Fortrea (1 mentions)
Anti egfr antibody, by Cell Signaling Technology (1 mentions)
Epidermal growth factor (egf), by Merck Group (1 mentions)
4 protocols using 647 secondary antibodies
1
Immunohistochemical Staining of Kidney Cells
2
Immunostaining of Oligodendrocytes
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Oligodendrocytes were stained as previously described [21 (link)]. Briefly, cells were washed with PBS, blocked with normal goat serum, permeabilized with Triton X-100, then incubated in primary antibody overnight at 4 °C. The primary antibodies used are listed in Table 1 . The following day, cells were incubated with corresponding AlexaFluor488, 568, or 647 secondary antibodies (1:1000, Invitrogen, Waltham, MA, USA) for 1 h at room temperature and co-stained with DAPI. Slides were cover-slipped with Prolong Gold Antifade Reagent (Invitrogen) and imaged using a Nikon-A1 Standard Sensitivity confocal microscope with NIS-Elements software. Four-to-eight images were randomly taken in each well, with 1–2 wells per mouse, and 2–5 mice per genotype.
3
Drosophila Larval Immunostaining Protocol
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Female larvae were dissected in Ca2+-free saline and fixed in 4% paraformaldehyde for 18–20 min or in cold methanol for 5 min (GluR antibodies). Larval body walls were incubated in primary and secondary antibodies overnight at 4°C. Body wall preparations were mounted in VectaShield (Vector Laboratories) for microscopic analysis. Larval brains were placed on poly-lysine–coated coverslips, dehydrated through ethanol series, cleared in xylenes, and then mounted in DPX (Sigma-Aldrich). The following antibodies were used: FITC- or Cy3-conjugated anti-HRP (Jackson ImmunoResearch Laboratories, Inc.) at 1:100, Texas red–X Phalloidin (Life Technologies) at 1:1,000, rabbit or chicken anti-GFP (Invitrogen) at 1:1,000, rabbit anti-pJNK (Cell Signaling Technology) at 1:1,000, rabbit anti-βgal (MP Biomedicals) at 1:1,000, rat anti-elav (Developmental Studies Hybridoma Bank) at 1:100, mouse anti-nc82 (Brp; Developmental Studies Hybridoma Bank) at 1:250, mouse anti-GluRIIA at 1:100 (Developmental Studies Hybridoma Bank), rabbit anti-GluRIIB (raised against ASSAKKKKKTRRIEK in Marrus et al. [2004] (link)) at 1:2,500, and rabbit anti-GluRIII (raised against QGSGSSSGSNNAGRGEKEARV in Marrus et al. [2004] (link)) at 1:1,000 (A. DiAntonio, Washington University, St. Louis, MO). Species-specific Alexa 488, 568, or 647 secondary antibodies were used at 1:500 (Invitrogen).
4
Receptor Agonist Synthesis and Antibody Procurement
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PAR1 peptide agonist (SFLLRN) and PAR2 peptide agonist (SLIGKV) were synthesized and purified by reverse-phase, high-pressure liquid chromatography at the Tufts University Core Facility (Boston, MA). Epidermal growth factor was purchased from Sigma-Aldrich (St. Louis, MO). Polyclonal anti-FLAG and anti-HA antibodies were purchased from Rockland Immunochemicals (Pottstown, PA). Anti-EEA1 antibody was obtained from BD Biosciences (East Rutherford, NJ). Monoclonal anti-PAR1 WEDE antibody was purchased from Beckman Coulter (Brea, CA). Polyclonal anti-ARRDC3, monoclonal anti-ALIX, and monoclonal anti-Ub (P4D1) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-EGFR antibody was obtained from Cell Signaling Technologies (Boston, MA). Monoclonal anti-HA antibody was purchased from Covance (San Diego, CA). Horseradish peroxidase–conjugated goat anti-rabbit and goat anti-mouse antibodies were purchased from Bio-Rad Laboratories (Hercules, CA). Alexa Fluor 488, 594, and 647 secondary antibodies were purchased from Invitrogen (Carlsbad, CA).
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