Thanks, Joe and Adam. I'm always paranoid about over tightening and warping some piece of the equipment but maybe I'm being a little too gentle. I'll make sure everything is nice and snug and see how it goes. Happy slicing... Nate Galbraith, Joe wrote:
Nathan: As indicated by others - first check that everything is tight as that is most often the issue - don't forget to check the adhesion of the specimen to the chuck as well. Also check the angle of approach to the blade as it could be too steep or too shallow. If everything is tight and properly adjusted, then you may have a retraction issue. If your cryostat retracts during the upstroke, the head may still be returning to the proper distance from the head to the specimen during the downstroke and hence you may see no section at first and then a gradually increasing thickness of section during the remainder of the downstroke. This can be due to two causes: 1) You can make this happen by operating the microtome at too high of a speed, if you slow down and the process improves then speed may be a factor; 2) Your microtome may be sticky and require maintenance (defrosting, cleaning, lubrication, etc.). The latter is best left to someone with extensive experience or a service rep. Good luck. Joe Galbraith University of Iowa [1][email protected] -----Original Message----- From: [2][email protected] [[3]mailto:[email protected]] On Behalf Of Adam . Sent: Thursday, August 27, 2009 4:53 PM To: Nathan Cramer Cc: [4][email protected] Subject: Re: [Histonet] uneven alternating sections on cryostat I was having this problem too, but I think I finally figured it out. There is a screw on our cryostat that attaches the chuck to the rest of the machine. This screws fits into a small hole in the chuck. Sometimes the screw isn't well set in the hole or is well set but for some reason comes loose and causes the chuck to wobble. For some reason, this causes your problem. Now I make sure that everything is tightly secured, and I rarely have this problem. Good luck, Adam On Thu, Aug 27, 2009 at 4:47 PM, Nathan Cramer [5]<[email protected]> wrote: Thanks, Robyn... I'll make sure the holder is clamped down. (sometimes its the little things...) Best Nate R J VAZQUEZ wrote: Nathan, It sounds like the blade holder is not secure enough or even in the tightness on each side. Hope this helps. Robyn > Date: Thu, 27 Aug 2009 16:44:11 -0400 > From: [[6]1][email protected] > To: [[7]2][email protected] > Subject: [Histonet] uneven alternating sections on cryostat > > When cutting PFA fixed, cryoprotected tissue on our cryostat, I > frequently find that every other section gets cut improperly. I'll get > one nicely cut section and then on the next pass I only get half of a > section. This cycle simply repeats over and over and I lose many slices. > It has been a while since our cryostat has been serviced, so I'm > wondering if this an operator error or a machine problem. I thought > maybe the tissue temperature hadn't settled properly, but the uneven > cutting still happens even when I let the tissue sit for 30 minutes in > the chuck holder. (cutting mouse spinal cord at -20C) > > On another note, if anyone has any tips for improving white/gray matter > contrast in frozen spinal cord sections stained with luxol fast blue, > I'd be very appreciative. I do defat the slices with chloroform and > differentiate with lithium carbonate but everything is usually either > very dark or very light. I am learning as I go (checking the archives > here often) so any help would be great. > > Thanks! > > Nathan Cramer > Neurobiology and Behavior > Cornell University > Ithaca, NY 14853 > > _______________________________________________ > Histonet mailing list > [[8]3][email protected] > [4][9]http://lists.utsouthwestern.edu/mailman/listinfo/histonet References 1. [10]mailto:[email protected] 2. [11]mailto:[email protected] 3. [12]mailto:[email protected] 4. [13]http://lists.utsouthwestern.edu/mailman/listinfo/histonet _______________________________________________ Histonet mailing list [14][email protected] [15]http://lists.utsouthwestern.edu/mailman/listinfo/histonet _______________________________________________ Histonet mailing list [16][email protected] [17]http://lists.utsouthwestern.edu/mailman/listinfo/histonet References 1. mailto:[email protected] 2. mailto:[email protected] 3. mailto:[email protected] 4. mailto:[email protected] 5. mailto:[email protected] 6. mailto:1][email protected] 7. mailto:2][email protected] 8. mailto:3][email protected] 9. http://lists.utsouthwestern.edu/mailman/listinfo/histonet 10. mailto:[email protected] 11. mailto:[email protected] 12. mailto:[email protected] 13. http://lists.utsouthwestern.edu/mailman/listinfo/histonet 14. mailto:[email protected] 15. http://lists.utsouthwestern.edu/mailman/listinfo/histonet 16. mailto:[email protected] 17. http://lists.utsouthwestern.edu/mailman/listinfo/histonet _______________________________________________ Histonet mailing list [email protected] http://lists.utsouthwestern.edu/mailman/listinfo/histonet
