[Histonet] Frozen brain slices

[ANJUM PARKAR]

Thanks Tina. I will keep the sucrose suggestion in mind for future harvests.
For now I need to figure out how to slice the already fixed frozen tissue. Will
try to equilibrate in the cryostat itself as against room temperature and see
how that goes. The knife angle is 13 degrees and recently sharpened and
thickness of slices 30 microns which I believe is standard for sectioning. I
have sectioned previously a lot so doing hands on is something I am familiar
with. But I did realize every instrument is different and while doing a
procedure it always needs to be optimized until it can become routine. Will let
you know how things go.
Anjum


On Sun, Nov 14, 2010 02:21 PM, "Montina Van Meter" <Montina.VanMeter <@t> pbrc.edu>
wrote:
>
Anhum,
>1. You MUST cryoprotect the fixed tissue in 15-30% sucrose (until it  
>sinks) prior to cutting frozen sections.
>2. Allow the -80C embedded tissue block to equilabrate to -20C in the  
>cryostat for 15-20min.
>3. Check the knife angle. Thickness?
>4. Change knife or sharpen and make sure all screws are tight.
>5. Freez/thawing of block is not a good idea (especially since it's  
>not cryoprotected). You are introducing ice crystal artifact.
>6. Check around your department (or Histology Core) to see if someone
> 
>can instruct you on using the cryostat. It's always better to actually  
>watch someone in addition to receiving written instructions.
>
>Good luck,
>Tina
>
>
>Sent from my iPhone
>
>On Nov 14, 2010, at 10:47 AM, "ANJUM PARKAR" <axp969 <@t> psu.edu>
>wrote:
>
>> Hello all,
>> I have been having some histology issues in terms of rat brain  
>> slicing the past
>> few months and hence looking for suggestions to fix them, having  
>> tried most of
>> what I know from my past training and running of ideas real fast now.
>> The following is the protocol I have used thus far:-1. Animal  
>> perfusion-a)Use
>> PBS first, b) Use 4%PFA next and c) Harvest brain right after
>PFA.
>> (Transcardial perfusion)2. Freeze brain by embedding in OCT
>compound  
>> using dry
>> ice and then storing at -80 until ready to slice. Alternatively some  
>> times the
>> -80 is used straight up with out dry ice freezing (still embedded in  
>> OCT).3.
>> From standard protocol I do not do two things-a) No post fixation  
>> once brain is
>> harvested and b) No sucrose. Reason being, I was taught that these  
>> two steps
>> might result in large holes in tissue.4.For slicing, we have the  
>> older cryostat
>> version (Real old version of CM3050S from Vibrotome-30 micron  
>> slices) which
>> uses a fixed knife for slicing and has a chamber temp control and  
>> one for the
>> specimen. The cryostat has not been serviced for a few years now.5.  
>> In attempt
>> 1-I take the brain out of the -80 once the chamber temperature comes  
>> to -20 and
>> mount the frozen mold on the chuck and attempt at slicing. Doing  
>> this I managed
>> to get a few slices (that too with cuts and ripples) and after
>which  
>> the tissue
>> just kept cracking and falling into pieces and hence could get no more
>> slices.6. In attempt 2-I thought the brain may be too cold and too  
>> hard
>> (perfusion and-80 freezing), so I defrosted the brain for 20
>mins at  
>> room
>> temperature before sticking into -20 cryostat. Between slices if I  
>> felt the
>> tissue was too cold, I put my thumb on the specimen to warm it up.  
>> Still no
>> luck.7. In attempt 3-I defrosted the brain completely to room  
>> temperature, took
>> the brain out of the cryogel embedding (thinking that the mold was  
>> way too hard
>> to cut through and hence cracking the tissue too) and attached it to  
>> chuck
>> directly and tried slicing. Still no luck at all this time around.
>> From what I have been reading online and from what I know, I  
>> primarily feel
>> these are issues related with temperature of the brain and that it  
>> is too cold
>> during cutting. I have tried different ways to bring the temperature  
>> down and I
>> still have no success. I have eight other brains frozen using the  
>> protocol
>> above and I really need to get slices out of them. Do you have any  
>> suggestions
>> for me? Also do you think I should make any changes to the existing  
>> freezing
>> protocol for the future brain harvesting?
>> APPenn State University,PA
>>
>>
>>
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