The mouse was anesthetized with overdosed isoflurane, and then perfused transcardially with 0.1 M phosphate buffer (PB) followed by 4% paraformaldehyde in PB. Brains were removed carefully and given an additional overnight postfix in 4% paraformaldehyde at 4°C, and then transferred to 30% sucrose in PB for 48 hr before sectioning. Coronal sections (35 μm) were cut on a cryostat (Leica CM1950), rinsed in 0.1 M PB, incubated in PB containing 0.5% sodium borohydride (30 min), rinsed, incubated in PB containing 0.5% H2O2 (15 min), and rinsed again. Primary and secondary antisera were diluted in PB containing 1% normal donkey serum (Jackson ImmunoResearch, West Grove, PA) and 0.3% Triton X-100 (Sigma). Sections were incubated with the primary antibody (polyclonal goat anti-choline acetyltransferase, AB144P, 1:200; Millipore Sigma) in the blocking solution overnight at room temperature. After rinsing, the sections were incubated with the secondary antibody (Alexa Fluor 594-conjugated donkey anti-goat IgG, 1:500; Abcam) for 90 min at room temperature, and mounted using Fluoromount-G mounting medium (SouthernBiotech). Fluorescence images were taken under an Olympus IX71 fluorescence microscope at 4x or 10x original magnification, using QImaging camera and MetaMorph Advanced software.
Alexa fluor 594 conjugated donkey anti goat igg
Manufactured by Abcam
Sourced in United States
Alexa Fluor 594-conjugated donkey anti-goat IgG is a secondary antibody that binds to goat immunoglobulin G (IgG) and is labeled with the red-fluorescent Alexa Fluor 594 dye. It is designed for use in immunofluorescence and other fluorescence-based applications.
Automatically generated - may contain errors
Lab products found in correlation
Normal donkey serum (nds), by Jackson ImmunoResearch (2 mentions)
Fluoromount g mounting medium, by Southern Biotech (2 mentions)
Cm1950, by Leica camera (2 mentions)
Ix71 fluorescence microscope, by Olympus (2 mentions)
Triton x 100, by Merck Group (2 mentions)
Ab144p, by Merck Group (2 mentions)
Alexa fluor 488 conjugated donkey anti rabbit igg, by Abcam (1 mentions)
Alexa fluor 594 conjugated goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Lsm 880, by Zeiss (1 mentions)
Alexa fluor 488 conjugated donkey anti goat igg, by Abcam (1 mentions)
4 protocols using alexa fluor 594 conjugated donkey anti goat igg
1
Immunohistochemical Visualization of Cholinergic Neurons
2
Immunohistochemical Visualization of Cholinergic Neurons
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The mouse was anesthetized with overdosed isoflurane, and then perfused transcardially with 0.1 M phosphate buffer (PB) followed by 4% paraformaldehyde in PB. Brains were removed carefully and given an additional overnight postfix in 4% paraformaldehyde at 4°C, and then transferred to 30% sucrose in PB for 48 hr before sectioning. Coronal sections (35 μm) were cut on a cryostat (Leica CM1950), rinsed in 0.1 M PB, incubated in PB containing 0.5% sodium borohydride (30 min), rinsed, incubated in PB containing 0.5% H2O2 (15 min), and rinsed again. Primary and secondary antisera were diluted in PB containing 1% normal donkey serum (Jackson ImmunoResearch, West Grove, PA) and 0.3% Triton X-100 (Sigma). Sections were incubated with the primary antibody (polyclonal goat anti-choline acetyltransferase, AB144P, 1:200; Millipore Sigma) in the blocking solution overnight at room temperature. After rinsing, the sections were incubated with the secondary antibody (Alexa Fluor 594-conjugated donkey anti-goat IgG, 1:500; Abcam) for 90 min at room temperature, and mounted using Fluoromount-G mounting medium (SouthernBiotech). Fluorescence images were taken under an Olympus IX71 fluorescence microscope at 4x or 10x original magnification, using QImaging camera and MetaMorph Advanced software.
3
Detecting Microglia in Spinal Cords
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Rats were transcardially perfused with ice-cold PBS followed by cold 4% paraformaldehyde (PFA). Spinal cords were then fixed in 4% PFA for 16 h. The next day, spinal cords were immersed in 30% sucrose-PBS for 3 days. Spinal cords were then embedded in paraffin. Five-micron-thick cross-sections were prepared and stained following the standard H&E staining protocol.
To detect the transferred microglia and endogenous microglia, the spinal cord sections were stained with the GFP antibody (1:1000, Abcam ab13970) and Iba1 antibody (10 μg/ml, Abcam ab5076) at 4 °C overnight. Alexa Fluor® 488-conjugated donkey anti-rabbit IgG (5 μg/ml, Abcam ab150073) and Alexa Fluor® 594-conjugated donkey anti-goat IgG (5 μg/ml, Abcam ab150132) were used as secondary antibodies to incubate the sections for 1 h at room temperature.
To detect the transferred microglia and endogenous microglia, the spinal cord sections were stained with the GFP antibody (1:1000, Abcam ab13970) and Iba1 antibody (10 μg/ml, Abcam ab5076) at 4 °C overnight. Alexa Fluor® 488-conjugated donkey anti-rabbit IgG (5 μg/ml, Abcam ab150073) and Alexa Fluor® 594-conjugated donkey anti-goat IgG (5 μg/ml, Abcam ab150132) were used as secondary antibodies to incubate the sections for 1 h at room temperature.
4
Histological Analysis of Mouse Brain
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At the end of the experiments, mice were anaesthetized with sodium pentobarbital and transcardially perfused with PBS. Brains were post-xed in 4% paraformaldehyde for 24 hours at room temperature and subsequently penetrated with 15% sucrose and 30% sucrose. Then brain tissues were sectioned into 6 µm thick coronal slices. For H&E staining and Nissl staining, brain slices were stained with hematoxylin and eosin, and toluidine blue, respectively. For immuno uorescence staining, slices were incubated with primary antibodies against NeuN (mouse, 1:200, Abcam, Temecula, CA, USA), GFAP (goat, 1:500, Abcam, Temecula, CA, USA) and Iba1 (goat, 1:200, Novus Biologicals, Littleton, CO, USA). Slices were then incubated with AlexaFluor 594-conjugated goat anti-mouse IgG (1:200, Carlsbad, CA, Invitrogen), AlexaFluor 488-conjugated donkey anti-goat IgG (1:200, Abcam, Temecula, CA, USA) and AlexaFluor 594conjugated donkey anti-goat IgG (1:200, Abcam, Temecula, CA, USA) secondary antibodies respectively.
Images were observed and taken randomly for each sample under a microscope (Olympus, Tokyo, Japan). Immuno uorescence images were observed with a confocal microscope (Zeiss LSM 880, Carl Zeiss, German).
Images were observed and taken randomly for each sample under a microscope (Olympus, Tokyo, Japan). Immuno uorescence images were observed with a confocal microscope (Zeiss LSM 880, Carl Zeiss, German).
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