Spinal cords were dissected and embedded in O.C.T., twenty-five-micron thick sections cut on a cryostat and mounted onto slides. Slides were dried at room temperature overnight, incubated in 0.1% Triton X- in PBS for 30 min, blocked in 4% BSA 1% Triton X- in PBS for 30 min, and incubated overnight at 4°C with goat anti-ChAT (1:100, Millipore Sigma-Aldrich, AB144P) and anti-STMN2 (1:100, rabbit anti-SCG10, Shin et al., 2012 (link)) in blocking buffer. The next day, slides were washed with 0.1% Triton X-3 times, incubated with Cy3 anti-goat (1:250, Jackson Immunoresearch, 705-166-147) and Alexa Fluoro-488 goat anti-rabbit IgG (H + L) (Invitrogen A21121) in wash solution for 2 h at room temperature, then washed in PBS and mounted in Vectashield with DAPI. Images of motor neurons in the lumbar spinal cord were taken on a Zeiss Axio Imager Z2 Fluorescence Microscope with ApoTome 2. Cell bodies were counted and averaged across 3 or more sections. For quantification of STMN2, motor neurons were identified by ChAT expression and the percent of STMN2+ motor neurons was calculated.
Cy3 anti goat
Manufactured by Jackson ImmunoResearch
Sourced in United States
Cy3 anti-goat is a secondary antibody labeled with the fluorescent dye Cy3. It is designed to detect and visualize goat primary antibodies in various immunoassays and microscopy applications.
Automatically generated - may contain errors
Lab products found in correlation
Ab144p, by Merck Group (2 mentions)
Alexa fluoro 488 goat anti rabbit igg h l, by Thermo Fisher Scientific (2 mentions)
Axioimager z2 fluorescence microscope, by Zeiss (2 mentions)
Anti rabbit cy3, by Jackson ImmunoResearch (1 mentions)
Anti cd40, by Bio-Rad (1 mentions)
Anti cpla2α, by Santa Cruz Biotechnology (1 mentions)
Anti cd206, by R&D Systems (1 mentions)
3 protocols using cy3 anti goat
1
Spinal Cord Motor Neuron Immunostaining
2
Spinal Cord Motor Neuron Immunostaining
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Spinal cords were dissected and embedded in O.C.T., twenty-five-micron thick sections cut on a cryostat and mounted onto slides. Slides were dried at room temperature overnight, incubated in 0.1% Triton X- in PBS for 30 min, blocked in 4% BSA 1% Triton X- in PBS for 30 min, and incubated overnight at 4°C with goat anti-ChAT (1:100, Millipore Sigma-Aldrich, AB144P) and anti-STMN2 (1:100, rabbit anti-SCG10, Shin et al., 2012 (link)) in blocking buffer. The next day, slides were washed with 0.1% Triton X-3 times, incubated with Cy3 anti-goat (1:250, Jackson Immunoresearch, 705-166-147) and Alexa Fluoro-488 goat anti-rabbit IgG (H + L) (Invitrogen A21121) in wash solution for 2 h at room temperature, then washed in PBS and mounted in Vectashield with DAPI. Images of motor neurons in the lumbar spinal cord were taken on a Zeiss Axio Imager Z2 Fluorescence Microscope with ApoTome 2. Cell bodies were counted and averaged across 3 or more sections. For quantification of STMN2, motor neurons were identified by ChAT expression and the percent of STMN2+ motor neurons was calculated.
3
Immunofluorescence Imaging of Microglial Markers
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Microglia were suspended in DMEM (2% FBS, 2 mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, and 12.5 U/ml Nystatin) and seeded on cover slips. The cells were fixed with ice-cold methanol for 3 min and then washed with HBSS. For immunofluorescence detection, the fixed microglial cells were incubated with the first antibody 1:50 in 5% BSA/PBS (anti cPLA2α (Santa Cruz Biotechnology, CA, USA), anti CD40 (Serotec, Cambridge, UK), anti CD206 (R&D Systems, Minneapolis, USA) Serotec, Oxfordshire, UK) for 90 min at room temperature. The cells were washed three times in HBSS and incubated with Cy3 anti-rabbit, DyLight anti-rabbit, and Cy3 anti-goat (1:50 in 5% BSA/PBS; Jackson ImmunoResearch Laboratories, Inc., PA, USA) for 60 min at room temperature. The cells were washed three times in HBSS, and the nuclei were stained with DAPI. Then, final wash was performed and the cells were taken to fluorescence microscope analysis (Olympus, BX60, Hamburg, Germany).
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