Exploring the functional neuroanatomy of neuronal nicotinic acetylcholine receptors

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 from here). 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 (download here)
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 here)
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) (download here)
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 (download here)
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 Molecular Probes and Biotium. 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 (download here)
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 analysis.

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.