Mice were intracardially perfused with ice-cold phosphate buffered saline (PBS) and 4% paraformaldehyde (PFA)/PBS (wt/vol, pH 7.4) after anesthesia by i.p. injection of Nembutal sodium solution (50 μl of twofold dilution in PBS of pentobarbital sodium 50 mg/ml; Lundbeck). Brains were removed from mouse heads, and postfixed for 24 h in the same fixative (4% PFA). After cryoprotection in 30% sucrose/PBS (wt/vol, pH 7.4) for 2–3 days incubation, brains were cut into coronal brain sections with a microtome (with 30 μm thickness). In immunostaining procedures, brain sections were blocked with 10% donkey/goat serum (sigma/Aldrich)/PBS plus 0.3% Triton X-100, and then incubated with indicated antibodies (4° C, overnight). After three washes (10 min each) with PBS, floating brain sections were incubated with corresponding secondary antibodies conjugated with fluorescent dyes. Images were obtained using a confocal microscope (Zeiss Confocal LSM 880 with Airyscan) and digital microscope (Keyence VHX 7000, Japan).
Donkey goat serum
Manufactured by Merck Group
Donkey/goat serum is a common laboratory reagent used in various biochemical and immunological applications. It is derived from the blood of either donkeys or goats and contains a complex mixture of proteins, antibodies, and other biomolecules. The serum can be used as a supplement in cell culture media or as a blocking agent in immunoassays to prevent non-specific binding.
Automatically generated - may contain errors
Lab products found in correlation
Triton x 100, by Merck Group (2 mentions)
Nucblue, by Thermo Fisher Scientific (2 mentions)
Donkey goat serum, by Thermo Fisher Scientific (2 mentions)
Vhx 7000, by Keyence (1 mentions)
Paraformaldehyde (pfa), by Electron Microscopy Sciences (1 mentions)
Fluoromount medium, by Merck Group (1 mentions)
Ab2480, by Abcam (1 mentions)
Igg free bsa, by Jackson ImmunoResearch (1 mentions)
5 protocols using donkey goat serum
1
Mouse Brain Tissue Preparation and Immunostaining
2
Pluripotency and Lineage Characterization of iPSCs
3
Pluripotency and Lineage Characterization of iPSCs
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Undifferentiated and differentiated iPSCs (passage 15) were assessed for pluripotency and lineage-specific markers by immunofluorescence staining. Cells fixed in 4 % paraformaldehyde (15 min, room temperature) were washed with DPBS, incubated with a permeabilization-blocking buffer composed of 0.1 % Triton X-100 (Sigma-Aldrich) and 2.5 % donkey/goat serum (Sigma-Aldrich) in DPBS (60 min, room temperature), followed by primary antibodies (Table 2 ) in the same buffer (overnight, 4°C). Following DPBS washes, cells were incubated with secondary antibodies (Table 2 ) in DPBS supplemented with 2.5 % donkey/goat serum (60 min, room temperature), washed more with DPBS, and counterstained with NucBlue (Thermo Fischer Scientific) (15 min, room temperature) before visualization using a Revolve Fluorescent Microscope (Echo Laboratories).
4
Immunofluorescence Staining of Cytoskeletal Proteins
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Cells were washed, fixed in 4% PFA (Electron Microscopy Science) for 10 min at 4°C, rinsed with PBS, permeabilized with a solution of 0.5% Triton X-100 in PBS for 5 min, incubated for 5 min with 5mM NH4Cl, and blocked in PBS containing 1% BSA (Jackson Immunology) and 1% goat/donkey serum (Sigma-Aldrich) for 30 min at RT. Cells were stained with phalloidin-Alexa Fluor 550 (Molecular Probes), mouse anti–β-catenin (CM1181; ECM Biosciences), rabbit anti–myosin light chain phospho S20 pMLC (ab2480; Abcam) primary antibodies (1 h in blocking buffer at RT), followed by incubation with the appropriate Dylight 480 or 650 secondary antibody (Thermo Fisher Scientific) for 45 min at RT. Coverslips were mounted in Fluoromount medium (Sigma-Aldrich), and fluorescent images were acquired on confocal microscopes (LSM 780 or 710; Zeiss) with a 63×/1.4NA oil-immersion objective.
5
Immunocytochemistry for Subcellular Imaging
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Cells were fixed in 4% PFA for 10 minutes at RT. Fixed cells were then blocked and permeabilized with the appropriate buffer (10% goat/donkey serum [Sigma‐Aldrich], 0.5% Triton‐X‐100, 2.5% IgG‐free BSA [Jackson Labs]) in PBS for 1 hour at RT. Cells were then incubated with primary antibody overnight at 4°C with agitation in blocking/permeablizing buffer without Triton‐X‐100. Next day, cells were washed thrice and incubated with PBS‐diluted secondary 1 hour at RT in darkness. Cells were then incubated for 30 minutes with 1:1,000 dilution 4′,6‐diamidino‐2‐phenylindole (DAPI; Sigma‐Aldrich) and phalloidin (PL) counterstain (Life Technologies), and imaged via confocal microscopy (LM700, Zeiss).
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