Protocol for Image processing and analysis of VPS13D recruitment to mitochondria
By onThis protocol details the image processing and analysis of VPS13D recruitment to mitochondria as it was performed in https://doi.org/10.1083/jcb.202010004. The first part details the analysis of acute optogenetic recruitment, and the second part of basal recruitment under different conditions.
Organelle isolation from Mouse Embryonic Fibroblasts (MEFs) stably expressing organelle tags for subsequent immunoblotting or proteomic analysis
By onWe describe here a method to perform rapid isolation of intact organelles (including lysosomes and Golgi) from mouse embryonic fibroblasts stably expressing an organelle tag (TMEM192-3xHA, or LysoTag, and TMEM115-3xHA, or GolgiTag). First, cells are broken using a ball-bearing cell breaker, leading to plasma membrane rupture, while lysosomes and Golgi remain intact. Then, the cell homogenate is incubated with anti-HA magnetic beads to allow for immunopurification of HA-tagged lysosomes or Golgi in less than 15 minutes. The organelles purified using this method are highly enriched, intact, contaminant-free and, depending on solubilisation buffer, can be used for various downstream applications, including immunoblotting analysis and mass spectrometry proteomic analysis (as described here), but also metabolomic or lipidomic analysis. This protocol can be adapted to isolate organelles from commonly cultured cells, such as HEK293 and A549 cells, that express an organelle tag.
Statistical analysis
By onThis protocol describes the statistical analysis applied to the quantifications in Chang et al. 2021 SA paper.
Neuromelanin-positive Neuron Density in Substantia Nigra Image Analysis
By onThe protocol covers the steps to measure neuromelanin-positive neuron density in substantia nigra using image analysis tools, including NZConnect (Hamamatsu), a web-based whole-slide image (WSI) viewer, Cellpose, and QuPath.
Chemical degradation and determination of pheomelanin and eumelanin markers
By onThis is protocol involving chemical oxidation and reduction methods followed by high performance liquid chromatography (HPLC) detection of markers for pheomelanin and eumelanin.
Immunohistochemistry of Human Brain Striatum: ciliation of cholinergic neurons and astrocytes
By onThe authors present a method to identify the same brain region in human postmortem tissue to evaluate ciliary status. The striatum is identified morphologically and also by immunofluorescence microscopy using anti-DARPP-32 antibodies.
Formalin Fixed Paraffin Embedded (FFPE) Tissue Preparation for Digital Pathology Quantification Using QuPath
By onQuPath is an open-source digital pathology analysis software. Here, it is used to quantify various misfolded proteins across anatomical regions.
Brain slice physiology and optogenetics
By onThe protocol outlines standard external solutions for electrophysiology experiments.
Image processing and 3D reconstruction
By onProtocol describing image processing and 3D reconstruction.
Sample vitrification and cryo-EM data acquisition
By onProtocol describing sample vitrification and cryo-EM data acquisition.
Free-floating Mouse Brain Immunohistochemistry
By onThis protocol enables immunohistochemical staining of murine tissue with superior penetration of the tissue by the reagents due to the free-floating approach.
Determination of free and protein-bound DA and NE and their metabolites and oxidation products by UPLC-MS/MS method
By onProtocol for the determination of free and protein-bound DA and NE and their metabolites and oxidation products by UPLC-MS/MS method.
Image processing to investigate mitophagy in HelaM and neurons
By onThis is a method for imaging cells and quantifying subcellular structures in the resulting data. We developed this protocol for assessing clearance of mitochondria involving OPTN and TBK1, however the method could be used for other applications.
Organelle isolation from mouse tissues expressing organelle tags
By onThe organelles purified using this method are highly enriched, intact, largely contaminant-free and can be used for various downstream applications, including immunoblotting analysis and proteomic analysis, but also lipidomic or metabolomic analysis.
A computational pipeline to quantify primary cilia in mouse embryonic fibroblasts with CellProfiler
By onThis protocol works with .czi format images which are acquired using a Zeiss laser scanning confocal microscope and are maximum intensity projected.
