INSTITUTE OF PHYSIOLOGY

Methods

Molecular biology:

  • cloning of GFP fusion proteins
  • introduction of mutations into proteins under examination

Cell culture:

primary:

  • brain slice culture - neuronal microcultures
  • transfection by calciumphosphate precipitation
  • gene gun transfection of slices

secondary:

  • Polyethylenimine (PEI) transfection
  • other

genegun
gene gun for transfection of brain slices

 

membrane
Plasma membrane targeting of a GFP labeled receptor tyrosine kinase in a secondary cell line (PC12 cells)

microcul
Synaptic microcultures consisting of isolated neurons on a restricted glial "island". Electrophysiological recordings from such synaptic pairs facilitate analysis of synaptic events

Electrophysiology:

setup
setup for fluorescence imaging and electrophysiology in brain slices

  • patch clamp recordings examination of transmitter
  • activated ion channels by a fast superfusion
  • system patch clamp recordings in acute
  • hippocampal and neocortical slices

 

Imaging:

visual
Visualization of dendritic spines and opposing presynaptic boutons in living neurons: dendritic segment of a GFP expressing hippocampal neuron (green) stained with the live presynaptic marker FM4-64

  • confocal microscopy
  • time lapse video microscopy of the dynamics of GFP fusion proteins in living cells
  • time lapse recordings of GFP fusion protein secretion by means of measuring the decrease of intracellular fluorescence
  • determination of presynaptic activity by kinetic measurements of the uptake and release of FM styryl dyes
  • immunocytochemical double and triple stainings
  • analysis of dendritic branching and spines in GFP expressing neurons

 

neuron
Hippocampal neuron in culture expressing BDNF-GFP. BDNF-GFP containing vesicles are targeted into neuronal processes

triple
Segment of a dendrite of a BDNF-GFP expressing neuron at high magnification, costained with the live synaptic marker FM4-64. Posthoc immunocytochemistry of the same segment identifies the dendritic marker MAP2

 

Last Modification: 14.05.2019 - Contact Person:

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