success rate of patching old animals

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EGC
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success rate of patching old animals

Dear fellows:
I'm doing a project that needs to be done with adult (> 40 days) rats and the success rate is very low.   I patched about 1 cell from 1 animal on average but only 1/3 cells can last long enough to give drugs. I kept having re-sealing problems. I tried many methods from other papers and wrote to those labs. They gave me all kinds of suggestions and tips. Still I got success some days and failure other days. I know the success rate on old animals is low but have no idea how good it can be. My boss kept blaming me for not getting good result contineously. Wheneve I didn't succeed I have to make some change, otherwise she would critisize me not making any effort. I'm reluctant to change my protocol too much because it still works (not as well as expected though).
Can anybody who is doing similar experiment give me a hint? If the success rate is as low as what I got now, I'll stay on my current method. Otherwise I need to figure out what's wrong and improve it. Thanks a lot.

vilsy
vilsy's picture
Hi there, I know what it

Hi there, I know what it feels to be in such a situation. I am assuming u r doing in vitro work and not in vivo. Working with older animals is a big pain for multiple reasons:
1. The quality of the tissue might not be so good : With older animals, I know one of my professors recommending perfusing the rat with ice cold ACSF before reoving the brain and sectioning. The tissue is healthier this ways. I have never tried it myself though
2. The resistances of the pipettes: These are critical. I would recommend keeping the tip size about 1.5-2um and resistance around 2-4MOhms. The smaller the better. This would make it easier to get a lock onto cells.

EGC
EGC's picture
Thank you, vilsy. I tried

Thank you, vilsy. I tried pipette with resistance around 3 but it is very hard to get seal. About perfusing rats, can you recommend any paper doing this way? If I can't do better with all other methods, I'll try it. Thanks a lot.

jilli
jilli's picture
In animal slices, specially

In animal slices, specially old ones, everyhting should be right. Starting from the beginning, if you work in a lab that uses fixatives, you must make sure that not even a drop of formalin enters your glassware, instruments. Autoclave glassware often. If you get toxic chemicals splashed to your bottles, even washing and autoclaving wont help. 
I have had success using surgery solution with no sodium (replacing it with sucrose) and high magnesium around 2 micromol/l. The surgery, should be fast, cold (although I have seen that too cold makes it worse). You must make sure to measure all salts very acurately. I have heard osmolarity makes a huge difference in cell servival.
Do you have access to Ketamine/xylasine anesthesia? It may help reducing initial spread of excitation while slicing.   
What vibratome are you using? Are you using a commercial holding chamber?
 

EGC
EGC's picture
jilli wrote:

jilli wrote:

In animal slices, specially old ones, everyhting should be right. Starting from the beginning, if you work in a lab that uses fixatives, you must make sure that not even a drop of formalin enters your glassware, instruments. Autoclave glassware often. If you get toxic chemicals splashed to your bottles, even washing and autoclaving wont help. 
I have had success using surgery solution with no sodium (replacing it with sucrose) and high magnesium around 2 micromol/l. The surgery, should be fast, cold (although I have seen that too cold makes it worse). You must make sure to measure all salts very acurately. I have heard osmolarity makes a huge difference in cell servival.
Do you have access to Ketamine/xylasine anesthesia? It may help reducing initial spread of excitation while slicing.   
What vibratome are you using? Are you using a commercial holding chamber?
 

 
Thank you for the reply. I use isoflurane to anesthesize animals. The vibratome is Microm HM 650V from Germany. Chamber is from warner instrument. THe Mg in my cutting solution is 4mM, sometimes even 7mM. All my stuffs are separated from others. I may autoclave my glassware.