Protocol Archive

This protocol details the preparation of free floating coronal mouse brain sections.

This protocol details the mouse stereotaxic injections into the mouse brain.

Using the Rubicon RH - Rab7 crystal structure as a template (6wcw).

Labels sites of autophagosome generation.

Following treatment with antibodies/genetic methods of introducing fluorescent tags.

1:1 binding of Rubicon and Rab7.

Production of pseudotyped virus an subsequent transduction.

Traditional in vitro pulldown.

Pulse-chaseable method for quantifying autophagy flux.

Phosphorylation of Rab7 at S72 using TBK1.

Method of quantifying in vitro binding.

Affinity purification followed by size exclusion chromatography.

Autophagosome live cell imaging for quantification of PINK-PARKIN mitophagy.

Plasmid construction via restriction enzyme digest and ligation.

Traditional Western blotting technique.

Growth in DMEM supplemented with FBS and antibiotics.

The authors describe their method to isolate peripheral blood mononuclear cells (PBMCs) and perform rapid isolation of intact, tag-less lysosomes from PBMC homogenates using ball-bearing cell breakers and anti-TMEM192 -antibody coupled to magnetic beads.

This protocol can be used to immunoprecipitate lysosomes from commonly cultured cells such as mouse embryonic fibroblast, HEK293, and A549 cells expressing the LysoTag.

Methods detailing the recording setup using both electrophysiological (Ephys) and fast-scan cyclic voltammetry (FSCV) are described.

This protocol describes how the authors prepare human iPSC-derived cells, that have been differentiated into cortical neuronal progenitors, for transplantation into the brain of immunocompromised athymic mice. Neuronal progenitors mature within the mouse brain and are used to study the pathogenesis of Parkinson’s disease.