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.vanme...@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" <axp...@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.vanme...@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" ><axp...@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 >> >> >> >> _______________________________________________ >> Histonet mailing list >> Histonet@lists.utsouthwestern.edu >> http://lists.utsouthwestern.edu/mailman/listinfo/histonet >> >> >> >> >> >> >> >> _______________________________________________ >> Histonet mailing list >> Histonet@lists.utsouthwestern.edu >> http://lists.utsouthwestern.edu/mailman/listinfo/histonet > > > -- Anjum Parkar Doctoral Candidate Department of Bioengineering, Penn State University, 206 Hallowell Building, University Park, PA 16802. _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet