[Histonet] Frozen brain slices

ANJUM PARKAR axp969 <@t> psu.edu
Tue Nov 16 17:55:27 CST 2010

Thanks Tina. I will try that for the already frozen brains. Also I did some
slicing today with some of my frozen brains and kept the block in the cryostat
till it came to temperature and as compared to before I think I can say that
the slices came out pretty well. I guess the temperature is the trick here.
Well I am going to run some stains on them and see how they turn out and then I
can say for sure that I have a good working protocol hopefully. These slices
are for slide mounting not free floating. I do use the anti-roll plate and I
did realize that it was a little off in its angle with respect to the knife and
hence one another issue that needed fixing. Will let you on my progress.
Thanks Liz for letting me know. Have you tried the Fluro Jade? The original, B
or C?

On Tue, Nov 16, 2010 05:33 PM, "Montina Van Meter" <Montina.VanMeter <@t> pbrc.edu>
>The post-fix and sucrose are for the new harvests.  
>Do you free-float the sections or mount them on slides?  
>Make sure you allow the -80C block of tissue to sit in the cryostat for about
>20 min. to allow the block to come up to the -20C temperature of the cryostat
>chamber.  There is no such thing as a "too hard block".  Allowing it
>to become the same temperature as the inside of the cryostat will solve that
>Warming up the block after freezing, than freezing it down again, is an
>absolute no-no.  This will also cause freeze/thaw artifact.  
>Are you using an anti-roll plate when sectioning on the cryostat?  That might
>help flatten out the sections as they come off the knife edge.  
>If you haven't already frozen the other brains I would place them in 20%
>sucrose/PBS overnight.  The next morning rinse off the sucrose with PBS for
>about 30 min.  Freeze the brain on dry ice in the OCT.  If you have already
>frozen all of the brains, you might take one of them and thaw it out.  Place it
>in the sucrose overnight and see if that helps with the cutting.  It can't
>hurt, since you are already having trouble getting sections.  
>Good luck,
>Montina J. Van Meter
>Lab Manager
>Autonomic Neuroscience
>vanmetmj <@t> pbrc.edu
>-----Original Message-----
>From: histonet-bounces <@t> lists.utsouthwestern.edu
>[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of ANJUM PARKAR
>Sent: Monday, November 15, 2010 9:24 AM
>To: Histonet <@t> lists.utsouthwestern.edu
>Subject: Re: [Histonet] Frozen brain slices
>Thanks Tina. I will keep in mind the thickness for staining purposes that is
>once the slicing protocol gets optimized. So we use 200-300 ml of both PBS and
>PFA for our perfusion. So the post fix and sucrose treatment you are
>suggesting, are you suggesting for the new harvests alone which I will be doing
>or the existing frozen perfused ones?
>On Sun, Nov 14, 2010 10:35 PM, "Montina Van Meter"
><Montina.VanMeter <@t> pbrc.edu>
>>How much PBS and Paraformaldehyde do you put through the (rat or  
>>mouse). I flush a perfuse our rats with 150-200ml. of PBS and 500ml.  
>>of Para. Postfix for 2 hours and place in 20% sucrose overnight until  
>>the tisue sinks to the bottom of the vial.
>>The morphology of your tissue is going to be compromised due to freeze  
>>artifact (lack of cryoprotectant) and there will be holes in your
>>Actually, 30um thick sections are considered quite thick and are  
>>typically used for free floating techniques. Sections that are mounted  
>>on slides before IHC staining are much thinner (3-10um).  I
>>cut my tissue between 30-40um and manually free float the sections for  
>>IHC or IF.
>>Sent from my iPhone
>>On Nov 14, 2010, at 9:05 PM, "ANJUM PARKAR"
><axp969 <@t> psu.edu>
>> 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
>> 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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>Anjum Parkar
>Doctoral Candidate
>Department of Bioengineering,
>Penn State University,
>206 Hallowell Building,
>University Park, PA 16802.
>Histonet mailing list
>Histonet <@t> lists.utsouthwestern.edu

Anjum Parkar
Doctoral Candidate
Department of Bioengineering,
Penn State University,
206 Hallowell Building,
University Park, PA 16802.

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