Thoracic ganglia were dissected in 1× phosphate‐buffered saline containing 0.05% Triton‐X‐100 (PBT) and then fixed overnight at 4°C in 2% paraformaldehyde (EM Sciences) in PBT. They were washed five times for 10 min in 0.1% PBT, blocked for 1 h in 0.1% PBT with 0.5% bovine serum albumin and 5% normal goat serum and then incubated with primary antibodies overnight at 4°C. They were then washed, blocked and incubated with secondary antibodies overnight at 4°C. Washed tissues were mounted on glass slides in Vectashield (Vector Laboratories), small pieces of broken coverslips served as posts, covered with a coverslip and sealed. Primary antibodies were rabbit anti‐DopR (1:1250; Kong et al.2010 ) and mouse anti‐TH (1:100, Immunostar), and secondary antibodies were goat anti‐rabbit Alexa Fluor 488 and goat anti‐mouse Alexa Fluor 594 (Life Technologies). The tissues were imaged on a Nikon Eclipse Ti C1 Confocal System using 1.15 µm steps and ×40 oil‐immersion lens. Quantification of signal intensity was performed using Image J (version IJ 1.46r) (http://imagej.nih.gov/ij/ ).
Eclipse ti c1 confocal system
Manufactured by Nikon
The Eclipse Ti C1 Confocal System is a high-performance microscope system designed for advanced confocal imaging. It features a modular design that allows for customization and integration with various accessories and imaging techniques. The system provides precise control over illumination and detection, enabling users to capture detailed, high-resolution images of biological samples.
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2 protocols using eclipse ti c1 confocal system
1
Immunostaining of Thoracic Ganglia Neurons
2
Immunostaining of Adult Brain Tissue
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Adult brains were dissected in 1 × PBS containing 0.05% Triton-X 100 (PBT), and then fixed overnight at 4° in 2% paraformaldehyde (EM Sciences) in PBT. They were washed for 5 × 10 min in 0.1% PBT, blocked for 1 hr in 0.1% PBT with 0.5% BSA and 5% normal goat serum, and then incubated with primary antibodies overnight at 4°. They were then washed, blocked, and incubated with secondary antibodies overnight at 4°. Washed tissues were mounted on glass slides in Vectashield (Vector Laboratories), small pieces of broken coverslips serving as posts, covered with a coverslip, and sealed. Primary antibodies were rabbit anti-DopR (1:1250, Kong et al. 2010 (link)), mouse anti-CD2 (1:50, Pierce), and mouse anti-Cherry (1:200, Biorbyt). Secondary antibodies were goat anti-rabbit Alexa Fluor 488 and goat anti-mouse Alexa Fluor 594 (Life Technologies). The tissues were imaged on a Nikon Eclipse Ti C1 Confocal System using 1 μm steps and a 40 × or 60 × oil-immersion lens.
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