Isolation and processing of embryonic and postnatal brains
By savannah onThis protocol describes: -Harvesting embryos from females from embryonic day 10 to embryonic day 18 -Brain isolation of embryonic (e10-e18) and postnatal brains (p0-p30) -Processing and freezing of embryonic and postnatal brains.
Immunofluorescence staining for postmortem mouse brain tissue
By savannah onThis is a basic protocol for staining mouse brain tissues using immunofluorescence and immunohistochemistry techniques.
Genotyping mice from ear clips
By savannah onThis protocol describes the genotyping procedure from ear clip samples. This includes a general PCR protocol for primers with annealing temperatures in the range 55-70 degrees C. For other primers, the thermal cycling should be adjusted.
Endosome isolation
By savannah onSubcellular fractionation to isolate early and late endosomes (EEs and LEs) by performing a series of centrifugation steps.
Create mask for axonal quantification analysis with FIJI
By savannah onThis protocol describes how to create a quantified mask from an image of fluorescence-tagged axonal projections using FIJI/ImageJ software.
Catalepsy test (Bar test)
By savannah onThe catalepsy test (bar test) was developed to test motor coordination and motor impairments.
Adhesive Removal Test to assess sensorimotor deficits in parkinsonian mice
By savannah onThis behavior is used to assess fine motor movements in a mouse Parkinsonian model. It checks for correct paw and mouth sensitivity (time-to-contact) and correct dexterity (time-to-remove).
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. Watch an interview about this protocol with Jonathan Breiter, MSc.
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.