Protocol for Preparation and Immunostaining of Vibratome Sections

Transcardial perfusion;

This procedure is typically performed on rats or mice. Be sure to get local approval from the relevant authorities before you attempt to do this. The procedure requires the use of drugs which are likely to require special purchase and regulatory steps in your country. Also the surgery is not trivial, if you have never done this before you should get help from someone who is an expert. The animal is first deeply anesthetized, and the heart exposed by opening the chest cavity. If you do this incorrectly you obviously may cause the animal great paom/

For mice, 50mL of fresh 4% paraformaldehyde (PFA) in PBS is injected into the ventricle using a syringe and circulated through the brain. For rats a larger volume of fixative is pumped into the heart typically using a mechanical pump. The brain is then extracted, and post-fixed overnight by submerging in a beaker containing more 4% PFA in PBS at 4°C. The next morning PFA is replace with PBS and the brain is stored at 4°C before cutting.

Vibratome sectioning;

the Vibratome is a popular sectioning device manufactured by Leica Biosystems which uses a vibrating razor blade to cut relatively thick sections of tissues. The tissues in general must be well fixed to cut easily. We typically mount CNS tissues on a platform with superglue and cut them directly. Smaller samples may be embedded in 1% agar. We usually cut 25-40μM sections of mouse and rat brain. Sections are floated off the specimen in PBS and are picked up and moved to multiwell plates with a paintbrush.

Free float immunolabelling;

1). We stain sections in 24 or 12 well multiwell plates. Non-specific binding is reduced by incubation in a blocking solution consisting of 10% normal goat serum in PBS containing 0.3% TX100. This is typically performed for 1hr at room temperature with gentle shaking.

2) Sections are then incubated in primary antibody prepared in blocking solution typically overnight at 4°C with gentle shaking.

3) Sections are washed in PBS 3 times for 5 minutes each time with gentle shaking at room temperature.

4) Sections are incubated in secondary antibody prepared in blocking solution for 1hr at room temperature with gentle shaking.

5) Sections are again washed in PBS 3 times for 5 minutes each time with gentle shaking.

6) Sections are carefully mounted using a paintbrush and coversliped for viewing. We routinely mount in Vectashield mounting medium with DAPI (Vector labs H-1200). The DAPI binds to DNA in the sections and so reveals nuclei of cells in blue on a typical fluorescence, confocal or 2-photon microscope.

Disclaimer; This protocol was constructed primarily to save time for busy researchers around the world. We hope you find it useful, and we are confident that it is reliable. However, we cannot be held responsible for any problems which may arise as a result of your use of this protocol.

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