Title: Standard Operating Procedure – Puncta colocalization analysis after VGlut1/Homer1 IF staining
Author: Rodrigo Sandoval Contreras, PhD and Nadia Haghbin, PhD
Date: 02/12/2025
Version: 1
Abstract: This protocol outlines a workflow for quantifying synaptic puncta and their colocalization following VGLUT1/Homer1 immunofluorescence staining in mouse brain tissue. The analysis combines high-resolution confocal microscopy with computational tools to achieve accurate, reproducible measurements. Images are processed and prepared for machine-learning-based segmentation using ilastik, which classifies synaptic puncta against background. Subsequently, SynBot, an open-source image analysis software, integrates these predictions to detect puncta and calculate colocalization metrics across experimental groups. This approach enables systematic, automated quantification of presynaptic and postsynaptic structures, providing robust datasets for studies of synaptic organization and connectivity.
DOI: coming soon
Link to Full SOP: mPCA-vglut1h1-v1
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Version Notes
[DATE]: Version [#]
- Adjusted the After reward pause from 5s to 10s
- Reasoning is explained at the end of the SOP, but was based on fibre photometry recordings that demonstrated mice needed more time to baseline
- Included an additional formula for more data we can analyze: Premature Responses
- Added another method to run the stimulus duration probe
- Clarified details about the Inter Trial Interval
- Specifically, we made it clearer that the ITI is variable, rather than a constant duration between each trial