[Histonet] Frozen brain slices

[ANJUM PARKAR]

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?
Anjum

On Sun, Nov 14, 2010 10:35 PM, "Montina Van Meter" <Montina.VanMeter <@t> pbrc.edu>
wrote:
>
Anhum,
>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  
>sections.
>
>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 usually  
>cut my tissue between 30-40um and manually free float the sections for  
>IHC or IF.
>
>Tina
>
>
>Sent from my iPhone
>
>On Nov 14, 2010, at 9:05 PM, "ANJUM PARKAR" <axp969 <@t> psu.edu>
>wrote:
>
>> 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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--
Anjum Parkar
Doctoral Candidate
Department of Bioengineering,
Penn State University,
206 Hallowell Building,
University Park, PA 16802.