Protocol Archive

This protocol evaluates motor coordination in the rotarod assay in parkinsonian mice.

The pole test evaluates the motor coordination ability of a mouse by having them grasp a pole and then descend to its home cage.

The Open Field task is a simple sensorimotor test used to determine general activity levels, gross locomotor activity, and exploration habits in rodent models of CNS disorders.

This 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.

Intraperitoneal L-dopa injection in mice.

This is a basic protocol for staining mouse brain tissues using immunofluorescence and immunohistochemistry techniques.

This 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.

Subcellular fractionation to isolate early and late endosomes (EEs and LEs) by performing a series of centrifugation steps.

This protocol describes how to create a quantified mask from an image of fluorescence-tagged axonal projections using FIJI/ImageJ software.

The catalepsy test (bar test) was developed to test motor coordination and motor impairments.

This 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).

While this protocol focuses recordings from striatum with GCaMP, it can be easily modified to record from other brain regions and with other fluorescent reporters.

This 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.

This protocol works with .czi format images which are acquired using a Zeiss laser scanning confocal microscope and are maximum intensity projected.

The 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 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.

This 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.

The 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.

The authors describe performing co-immunoprecipitation experiments in HEK293 cells using GFP-trap beads.

This 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).