To label cell surface GABAA receptors, primary neurons that were cultured on coverslips, were fixed with 2% paraformaldehyde in DPBS, blocked with goat serum for 0.5 h at room temperature, and labeled with 100 μL of appropriate anti-α1 (Synaptic Systems, catalog #: 224203), β2/3 (Millipore, catalog #: 05-474), or γ2 (Synaptic Systems, catalog #: 224003) antibodies (1:200) for 1 h without detergent permeabilization. Afterwards, they were incubated at room temperature with 500 μL (1:400) of Alexa 594-conjugated goat anti-rabbit antibody (ThermoFisher, catalog #: A11037), or Alexa 594-conjugated goat anti-mouse antibody (ThermoFisher, catalog #: A11032) for 1 h. Afterwards, cells were permeabilized with saponin (0.2%) for 5 min and incubated with DAPI (1 μg/mL) for 3 min to stain the nucleus. The coverslips were then mounted and sealed. For confocal immunofluorescence microscopy, an Olympus IX-81 Fluoview FV1000 confocal laser scanning system was used. A 60× objective was used to collect images using FV10-ASW software. Quantification of the fluorescence intensity was achieved using the ImageJ software from the NIH.
Alexa 594 conjugated goat anti mouse antibody
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Alexa Fluor 594-conjugated goat anti-mouse antibody is a secondary antibody used in immunoassays and other fluorescence-based applications. It is designed to bind to mouse primary antibodies, allowing for their detection and visualization.
Automatically generated - may contain errors
Lab products found in correlation
Alexa 594 conjugated goat anti rabbit antibody, by Thermo Fisher Scientific (3 mentions)
Anti α1, by Synaptic Systems (2 mentions)
Ix 81 fluoview fv1000 confocal laser scanning system, by Olympus (2 mentions)
Alexa 488 conjugated goat anti rabbit antibody, by Thermo Fisher Scientific (2 mentions)
Goat serum, by Thermo Fisher Scientific (1 mentions)
Goat serum, by Avantor (1 mentions)
Fluoromount g, by Avantor (1 mentions)
4 6 diamidino 2 phenylindole dapi, by Abcam (1 mentions)
Ox 42, by Abcam (1 mentions)
Confocal laser scanning microscope, by Olympus (1 mentions)
5 protocols using alexa 594 conjugated goat anti mouse antibody
1
Labeling GABAA Receptors in Neurons
2
Quantifying GABA Receptor Subunits in iPSC Neurons
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Neuron staining and confocal immunofluorescence microscopy analysis were performed as described previously 14 (link). Briefly, to label cell surface proteins, iPSC-derived GABAergic neurons on the chamber slides were fixed with 4% paraformaldehyde in DPBS for 10 min. We then blocked with 10% goat serum (ThermoFisher, catalog #: 16210064) in DPBS for 0.5 h, and without detergent permeabilization, incubated with 100 μL of appropriate primary antibodies against the GABAA receptor subunit (Synaptic Systems, Goettingen, Germany, catalog #: 224203) (1:250 dilution), subunit (Millipore, catalog #: 05–474) (1:250 dilution), or subunit (Synaptic Systems, Goettingen, Germany, catalog #: 224003) (1:250) diluted in 2% goat serum in DPBS, at room temperature for 1 h. Then the neurons were incubated with Alexa 594-conjugated goat anti-rabbit antibody (ThermoFisher, catalog #: A11037), or Alexa 594-conjugated goat anti-mouse antibody (ThermoFisher, catalog #: A11032) (1:500 dilution) diluted in 2% goat serum in DPBS for 1 h. Afterward, the chamber slides were mounted using fluoromount-G (VWR, catalog #: 100502–406) and sealed. An Olympus IX-81 Fluoview FV3000 confocal laser scanning system was used. A 60× 1.40 numerical aperture oil objective was used to collect high-resolution images using FV31S-SW software. The images were analyzed using ImageJ software 69 (link).
3
Immunohistochemical Analysis of Spinal Cord
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The lumbosacral enlargement was collected in mice of CON group and SR group, fixed in 4% paraformaldehyde, and dehydrated in 30% sucrose. The third to fifth lumbar vertebrae (L3–L5) were transversely cut into 25-μm-thick sections and mounted on glass slides. Sections were blocked and then incubated with primary antibodies against Iba-1 (1:700, Wako, Tokyo, Japan) and GFAP (1:500, Abcam, Cambridge, UK) or CD31 (1:500, Abcam, Cambridge, UK) overnight at 4°C. After washing with PBS, the sections were incubated with Alexa 488-conjugated goat anti-rabbit antibody (1:1000, Thermo Fisher Scientific, MA, USA) and Alexa 594-conjugated goat anti-mouse antibody (1:1000, Thermo Fisher Scientific, MA, USA) in the dark. Three spinal cord sections per animal were used for statistical analysis. The fluorescence intensities of Iba1, GFAP, and CD31 were measured using ImageJ software (NIH, Bethesda, MD, USA).
4
Immunohistochemical Analysis of Spinal Cord
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After general anesthesia was administered, mice were intracardially perfused with normal saline followed by 4% paraformaldehyde. The lumbar enlargements were removed, post-fixed with 4% paraformaldehyde and then dehydrated in 30% sucrose. Samples were cut into 20 µm sections using a freezing microtome. After washes with phosphate buffer saline (PBS), the sections were blocked with 10% goat serum containing 0.3% Triton X-100 and then incubated with the following primary antibodies in 10% goat serum overnight at 4℃: Iba1 (rabbit, 1:500, Wako, Japan), GFAP (mouse, 1:500, CST, USA), CB2R (rabbit, 1:100, Abcam, USA) and OX42 (mouse, 1:500, Abcam, USA). After washes with PBS, sections were incubated with secondary antibodies in 10% goat serum, including an Alexa 488-conjugated goat anti-rabbit antibody (1:3000, Thermo Fisher, USA) and Alexa 594-conjugated goat anti-mouse antibody (1:3000, Thermo Fisher, USA). Then, sections were washed with PBS, transferred to glass slides, air-dried and stained with 4’,6-Diamidino-2-Phenylindole (DAPI) (Abcam, USA). Images were captured using a laser-scanning confocal microscope (Olympus, Japan). Three sections from three mice from each group were analyzed.
5
Labeling Cell Surface GABAA Receptors
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To label cell surface GABAA receptors, primary neurons that were cultured on coverslips, were fixed with 2% paraformaldehyde in DPBS, blocked with goat serum for 0.5 h at room temperature, and labeled with 100 µL of appropriate anti-α1 (Synaptic Systems, catalog #: 224203), β2/3 (Millipore, catalog #: 05-474), or γ2 (Synaptic Systems, catalog #: 224003) antibodies (1:200) for 1 h without detergent permeabilization. Afterwards, they were incubated at room temperature with 500 µL (1:400) of Alexa 594-conjugated goat anti-rabbit antibody (ThermoFisher, catalog #: A11034), or Alexa 594-conjugated goat anti-mouse antibody (ThermoFisher, catalog #: A11037) for 1h. Finally, cells were permeabilized with saponin (0.2%) for 5 min and incubated with DAPI (1 µg/mL) for 3 min to stain the nucleus. The coverslips were then mounted and sealed. For confocal immunofluorescence microscopy, an Olympus IX-81 Fluoview FV1000 confocal laser scanning system was used. A 60× objective was used to collect images using FV10-ASW software. Quantification of the fluorescence intensity was achieved using the ImageJ software from the NIH.
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