On Jun 24, 7:41 am, Bill <connelly.b... from gmail.com> wrote:
> The principle behind the sucrose solution is simple. During strong
> insults to the brain, depolarization causes Cl- influx which
> eventually bursts the cell due to osmotic pressure. Further down the
> track, the calcium influx causes by the depolarization has activated
> cell death pathways. By removing Na+ and Cl- from the solution, you
> stop any depolarization and you stop and Cl- influx respectively. You
> can actually use a low sodium solution (Like Choline Cl-) because
> without the Na+ you can't get the depolarization in the first place.
> But weighing out sucrose is more pleasant than choline chloride.
>> See the original paper on Sucrose by George Aghajanian, where it was
> developed for making slices of the facial nuclei (not too far from
> where you are working)
>> Aghajanian GK, Rasmussen K.
> Intracellular studies in the facial nucleus illustrating a simple new
> method for obtaining viable motoneurons in adult rat brain slices.
> Synapse. 1989;3(4):331-8
>> I think who ever said they used 124mM sucrose made a mistake (I hope
> they did at least), you need to double the sucrose relative to
> NaCl... The solution I use is 248 sucrose, 3 KCl, 2 MgCl2, 1 CaCl2,
> 1.25 NaH2PO4, 26 NaHCO3, and 10 D-glucose. Some people use 10mM Mg2+
> and 0.1mM Ca2+ but I never found it made a difference.
>> I strongly recommend the sucrose solution, I expect you will find a
> vast improvement in your slices (where ever you cut your slices from
> in fact).
>> I also recommend incubating your slices at 35 degrees for half an hour
> to an hour after slicing. Another thing which I have found enhances
> the viability of some neurons is adding 1mM Na Ascorbate and 3mM
> Pyruvate (makes CA3 pyramidal, dentate granule and cerebellar granule
> neurons appear more healthy, but seems to have no effect on cortical
> neurons).
>> Finally, 20 days seems to be getting a little on the old side for such
> a mylin rich area. When I have worked in those kind of places, 21 days
> old was my absolute limit for finding viable cells. If at all possible
> I would work from 17 days old or younger.
>> All of that wont help too much with the problem of minigies. My only
> advise would be to take your time. With animals of this age, the
> decapitated brain seems to survive for a long time before you need to
> cut slices. Get you boss to buy you some ultra fine forceps, some
> moria spring scissors and a dissecting scope, and make sure you don't
> have any coffee before the dissection!
>> On Jun 19, 12:47 pm, wwt <wwt0... from gmail.com> wrote:
>>>> > Hi! everyone. Recently, I was cutting inferior olive sagittal slice in
> > rats (about 20d, female and male) and doing patch on these.
> > Unfortunately, the health of slice always was too bad. As you know,
> > the inferior olive is at very superficial position of ventral brain
> > stem. When I extract the brain stem, I feel it is too difficult to
> > avoid hurting the inferior olive. And there is a layer of meninge
> > adhering on the surface of inferior olive. It is almost impossible to
> > cut off meninges clearly in 20d rats during my operation. So the brain
> > stem is always extruded during cutting process. But if I clean the
> > meninges before cutting, it is ease to drag the tissue, even operation
> > carefully. How can I do for avoiding these?
> > Yosi Yarom's Research Lab use sucrose ACSF to cut inferior olive
> > slice. The concentration of sucrose is about 124mM in their cutting
> > solution( for example: J Neurophysiol 85:1686-1696, 2001.http://jn.physiology.org/cgi/content/full/85/4/1686#BIBL). And there
> > are no NaCl in cutting solution. But this solution is a kind of low
> > osmotic pressure, about 220 mOsm. I feel it is strange. Do you know
> > the principle or reason of it?- Hide quoted text -
>> - Show quoted text -
Thank you for your detaily answer. I will try more according your
advises.
A little question: What is the difference between Na Ascorbate and
Ascorbic acid? Can I use Ascorbic acid to replace the role of Na
Ascorbate?
