研究目的
To describe a novel dissection and recording technique for optical monitoring of staining and de-staining of lanceolate terminals in hair follicles of mouse ear skin, enabling study of vesicle recycling and its modulation.
研究成果
The novel pinna preparation provides a simple and effective method for optical monitoring of vesicle recycling in living lanceolate nerve terminals, allowing pharmacological and mechanical studies. It facilitates high-quality imaging with minimal preparation and is suitable for use in wild-type and genetically modified animals, offering insights into mechanosensory terminal function and modulation.
研究不足
The technique requires practice for optimal dissection and labeling; labeling is less reliable in larger mice (>25 g), and phototoxicity can damage terminals if excitation light is not minimized. Variability in labeling intensity and non-normal distribution of data necessitate careful statistical handling.
1：Experimental Design and Method Selection:
The protocol involves dissecting mouse pinnae, labeling nerve terminals with styryl pyridinium dyes, and imaging them using fluorescence microscopy to monitor dye uptake and release, reflecting vesicle recycling.
2：Sample Selection and Data Sources:
Adult mice (e.g., C57/Bl6J and MF1 strains) are used, with ears removed and prepared in saline.
3：List of Experimental Equipment and Materials:
Equipment includes scissors, forceps, insect pins, culture dishes, water bath, microscope with fluorescein filter set, digital camera; materials include saline, styryl pyridinium dye (e.g., FM1-43), Advasep-7, and pharmacological agents.
4：Experimental Procedures and Operational Workflow:
Steps include ear dissection, dye labeling with incubation at 30°C, washing to remove non-internalized dye, chelation with Advasep-7, and imaging with minimized light exposure to avoid phototoxicity.
5：Data Analysis Methods:
Intensity of labeled terminals is analyzed using annular regions of interest (ROIs) in image analysis software, with background subtraction and non-parametric statistical tests for comparisons.
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