Acute striatal or midbrain fiber photometry in head-fixed mice
By savannah onWhile this protocol focuses recordings from striatum with GCaMP, it can be easily modified to record from other brain regions and with other fluorescent reporters.
Free-floating mouse brain immunohistochemistry
By savannah onThis protocol enables immunohistochemical staining of murine tissue with superior penetration of the tissue by the reagents due to the free-floating approach.
A computational pipeline to quantify primary cilia in mouse embryonic fibroblasts with CellProfiler
By savannah onThis protocol works with .czi format images which are acquired using a Zeiss laser scanning confocal microscope and are maximum intensity projected.
Preparation of unilamellar liposomes
By savannah onThe authors present here a protocol for preparing liposomes that can be used to monitor binding of proteins to vesicles of various membrane curvatures. The authors used this to study the association of the PPM1H phosphatase with highly curved membranes due to its N-terminal amphipathic helix.
Gibson Assembly Cloning
By savannah onGibson assembly requires a vector backbone and one or more inserts that have been PCR amplified. The inserts should have 15-20 base pairs of overlap at ligation sites, so primers used to amplify the inserts should contain a tail overhang with this homology. The vector backbone should be linear.
Astrocyte isolation and culturing
By savannah onThis protocol details steps for preparing an astrocyte monoculture from mouse pups. From dissection to plated samples ready to be transfected, treated, lysed, or imaged, the protocol takes 20-25 days.
Cell lysis and gel electrophoresis for protein analysis of HeLa cells
By savannah onThe authors present multiple protocols used for biochemical analysis of protein expression and association. The authors’ studies demonstrated that NEMO recruitment to damaged mitochondria occurs in specific circumstances, and NEMO colocalization with p62 is also dependent on multiple factors.
Co-immunoprecipitation using GFP-trap
By savannah onThe authors describe performing co-immunoprecipitation experiments in HEK293 cells using GFP-trap beads.
Expression and purification of human NEMO (GST-GFP-NEMO)
By savannah onThis protocol describes how to express and purify human NEMO (IKK-γ) tagged N-terminally with GST and EGFP. The expression is performed with E. coli Rosetta pLysS cells. The protein is purified via a GST batch purification and gel filtration (SEC).
Fixation and imaging of HeLa cells after mitochondrial depolarization
By savannah onEctopic expression of p62/SQSTM1 often induces puncta formation and poor cell health due to p62’s proclivity to multimerize and form filaments. We noted modifying fixation protocol to visualize various endogenous ATG8 proteins, which are difficult to over-express in our system. Fixation techniques are a critical complement to studies in live cells.
Fixing hippo neurons to assess endogenous NEMO during oxidative stress
By savannah onThis protocol details how to assess endogenous NEMO during oxidative stress in fixed hippocampal rat neurons. Mitochondrial damage was induced by Antimycin A, endogenous NEMO was visualized with the commercially available anti-NEMO primary antibody (abcam), and mitochondria were visualized by a mito-targeted construct, Mito-SNAP.
HEK293 cell culture for co-immunoprecipitation experiments
By savannah onThe authors describe HEK293 cell culture for the purpose of co-immunoprecipitation experiments.
HeLa culture, transfection, and labeling of Halo-fusion proteins
By savannah onThe authors developed a protocol to investigate the role of the NEMO in Parkin-dependent mitochondrial clearance. Halo-fusion constructs, including NEMO and OPTN used in the study, allowed the authors to visualize the exogenously expressed proteins conjugated to chemical ligands in a variety of colors. This and the accompanying protocols were critical to the characterization of NEMO’s involvement in mitophagy.
Image processing to investigate NEMO recruitment and involvement in mitophagy and inflammatory signaling
By savannah onThe authors approached image analysis in a multitude of creative, effective ways, and importantly the authors maintained consistency of analysis within experiments in order to present the results with integrity and reproducibility.
Immunostaining of iPSC-derived neurons for quantification of synaptic proteins
By savannah onHere, we fix, permeabilize, and stain human iPSC-derived neurons for the purpose of observing and quantifying somal proteins of interest. For preceding culture of neurons, see "Protocol: Culture and transfection of iPSC-derived neurons for live-imaging of axonal cargoes."
Live imaging and analysis to investigate phase separation properties of NEMO during mitophagy
By savannah onThree standards of the field for determining phase separation are: (A) particles undergo fission and fusion like droplets of water, (B) individual puncta recover fluorescence after photobleaching, and (C) particles dissipate upon exposure to 1,6-Hexanediol. This protocol describes the authors’ approach to investigating standards B and C.
Live imaging to investigate mitophagy kinetics and NEMO recruitment in HeLa-M cells
By savannah onThe live imaging of NEMO occupancy on damaged mitochondria was a necessary complement to the authors’ parallel fixation studies. Their reporting of these results would not have been possible without real-time, live cell imaging.
Live-cell imaging for synaptic vesicle precursors in human iNeuron axons
By savannah onThe authors describe procedure and equipment used for live-imaging of synaptic vesicle precursors. This was performed using DIV21 human iPSC-derived excitatory glutamatergic neurons. Equipment and software used varied based on scheduled upgrades to microscopy equipment during the course of this study.
Microscopy-based bead protein-protein interaction assay
By savannah onThis protocol describes how to perform microscopy-based bead protein-protein interaction assay with GST- or mCherry-tagged proteins as baits and fluorescently-tagged proteins as preys. The protocol requires to have purified proteins and allows to monitor protein-protein interaction in an equilibrium state. The fluorescent signal can be quantified.
Gene editing of YIPF4 in hESCs V3
By savannah onThis protocol describes the creation of YIPF4 knockout cell lines in H9 hESC cells using CRISPR-Cas9. Associated with preprint: https://doi.org/10.1101/2022.12.06.519342