[Neuroscience] Re: How do you approach a neuron under DIC optic?
bilz0r at hotmail.com
bilz0r at hotmail.com
Tue Sep 6 22:15:02 EST 2005
Thanks for the tips,
The techneque you described for the production of brain slices is
exactly what we used to do when we made rat brain slices, but we had to
start using a sucrose based solution when we moved into the mouse.
We've got a Leica VT1000 coming, so I can try making thinner slices, so
I can get a better view at the neurons.
Have you ever pathced in cell culture? My supervisor has worked solely
in culture, and so the techneque he uses there is to poke the electrde
quite deep into the cell, as to get a massive (~20-50%)increase in
electrode resistance? How hard against the cell do you push? Would it
move a significant distance? What kinda increase in open-tip resistance
would you get?
> Slice health problems can also lead to a) bad seals and b) cells (but
> not the whole slice) moving around under positive pressure.
> If you notice that just the cells you're close to are going out of
> focus (or squeezing themselves out from under your pipette), and not
> cells far away, I would suspect that the tissue isn't very healthy.
> Also if it's hard to see things in general, if cells look either
> charcoal-ish or transparent, if there are obvious gaps between cells in
> places where there aren't supposed to be gaps (for example, the
> hippocampal CA1 or GC cell body layer) or if you cannot see clearly to
> depths of about 50 microns (many cell body widths down into the
> tissue), any of these are signs that your slices aren't happy.
> Nobody likes to think that their slices aren't healthy. But in case you
> suspect that, I would try the following:
> 1) Use a good vibratome. I hate the big green/black Camden ones, and I
> love the Leica VT1000.
> 2) Use a fresh blade every day, and use half of a double-edged blade
> rather than a single edged blade.
> 3) Wash the blade with acetone and then RINSE WELL with distilled water
> before putting on the slicer.
> 4) Make fresh ACSF every day, and make your solutions as cold as
> possible. We put ours in a -80 freezer for about 40 minutes until they
> form a 1 cm crust of ice all the way around the sides of the beaker.
> Then we break up the ice with a sturdy weighing spatula, scrape the ice
> off all the sides into the solution, then puree the whole thing with a
> hand-blender (has the consistency of a Marguerita when we're done, but
> too salty, especially after adding the tequila ;-] If your fingers do
> not ache from the cold during the dissection, it is probably not cold
> 5) Above all, absolutely minimize any pushing, pulling, stretching or
> any other kind of mechanical force on the brain and tissue during the
> entire procedure. Obviously there must be some, but take any steps you
> possibly can to make it is gentle as possible (AND FAST - brain should
> be out and mounted on the vibratome with 5-10 minutes after
> decapitation). Also, the less time the actual slicing takes, the
> better. I typically do about 10 hippocampal slices (5 from each
> hemisphere) in about 30 minutes, from the time of decapitation. Make
> sure evrything is well oxygenated all the time.
> 6) Clean the living crap out of everything (dissection tools, beakers,
> slicer, slice holding chamber, anything the comes in contact with a
> solution that comes in contact with the tissue) every day with
> distilled water ONLY - No soap. Pay special attention to the slice
> holding chamber. Run your bare finger around in it to see if the walls
> or the mesh (if there is one - there should be) are slimy. Slimy is
> very very bad.
> Some people like to use the saphire vibratome blades. We have never
> tried it, but have also never found it necessary to try. Some people
> also replace Na with sucrose, or add calcium channel/NMDA antagonists,
> etc. We have never found those to make any difference.
> Good luck,
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