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.
Alexa fluoro 488 goat anti rabbit igg h l
Manufactured by Thermo Fisher Scientific
Alexa Fluoro-488 goat anti-rabbit IgG (H + L) is a fluorescently-labeled secondary antibody used to detect and visualize rabbit primary antibodies. It binds to the heavy and light chains of rabbit immunoglobulin G (IgG) and is conjugated with the Alexa Fluor 488 fluorescent dye, which emits green fluorescence when excited.
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2 protocols using alexa fluoro 488 goat anti rabbit igg h l
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.
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