Exploring the functional neuroanatomy of neuronal nicotinic acetylcholine receptors
Ian Jones, PhD
On this page you can find a collection of immunolabelling
protocols commonly used in the lab. The protocols are saved as PDF files,
which require Adobe Acrobat Reader for viewing (can be downloaded free
As with most methods, these are not 'set in stone' and are ameanable to
tweaking here and there (different fixatives, buffers, detection systems,
etc). If there is anything that I/we do that is not covered here, let
me know and I will endeavour to rectify matters.
Immunohistochemisrty - 'ABC' method
This protocol describes a standard method for immunolabelling
vibratome sections using the HRP substrate diaminobenzidine (DAB). The
labelled sections can be viewed at the light microscope level or processed
for electron microscopy for subcellular analysis.
Immunofluorescence-cell cultures and tissue sections (download
Immunofluorescence (confocal) microscopy is used extensively within the
group for multiple labelling studies, often using three different primary
antibodies. The protocol outlines the basic labelling methods used with
cell cultures and tissue sections.
Synaptosomes (isolated nerve terminals)
Synaptosomes (essentially free-floating axon terminals) can be treated
very much like cell cultures. I've included them as a separate immunofluorescence
method as there are a few extra steps needed to stick them to slides.
Fluorescent bungarotoxin labelling
Alpha bungarotoxin is a snake neurotoxin with high affinity and specificity
for alpha 7 nAChR. Consequently it can be utilised as a tool for localising
alpha 7 nAChR in cell cultures and in tissue sections. Fluorescent conjugates
of alpha bungarotoxin are commercially available form companies such as
This file contains two methods. The first describes simple fluorescent
bungarotoxin single labelling of tissue sections. The second describes
a method for combining bungarotoxin labelling with immunofluorescence
(antibody) labelling. The methods can be easily adapted for cell culture
labelling, perhaps ommiting the permeabisation step.
Gold bungarotoxin - pre embedding
Recently we have characterised a 1.4nm gold conjugate of alpha bungarotoxin
(an alpha 7 specific nAChR toxin) for use in light and electron microscopy
(Jones et al., 2004, J Neurosci Methods; PDF
file). Here is a basic method for pre-embedding labelling using the
conjugate (could be used for any gold tagged marker).The protocol describes
embedding for electron microscopy, but is easily changed for light microscopy
Neurobiotin (download here)
Integrating electrophysiological studies with immunolabelling is an easy
way to generate more data from an experiment. During electrophysiological
recordings, neurones are filled with neurobiotin and then subsequently
fixed. This protocol describes how to detect the neurobiotin using immunofluorescence
and co-label with other proteins.
Osmium-free embedding for electron
microscopy (download here)
Osmium is often used as an additional fixative in preparing samples for
electron microscopy. However, osmium is detremental to antigenicity and
therefore only a few antibodies tend to work in post-embedding labelling
of osmium fixed tissue. To overcome this, Aldo Rustioni and Kris Phend
at the University of Noth Carolina have developed an osmium-free embedding
protocol which better retains tissue antigenicity (Phend et al., 1995,
J. Histochem. Cytochem. 43: 283-292). I am grateful to both Aldo and Kris
for sharing their protocol with me.
DAKO hadbook (download here)
OK, not exactly a lab protocol, but a very useful resource for those interested
in the theory and application of immunlabelling techniques.
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