Hi Heather, I can see why you’re having trouble. 30 micron sections are inherently unstable. Like paraffin, the thicker a section is the more difficult it is to cut. Plus since your practice samples are old they tend to be more brittle.
Try cutting at 5 microns and see what happens. Remember to let the samples warm up in the cryostat if they have been stored at -70 or -80 degrees. If they are too cold they are brittle too. I have not tried the tape method. We just place the sections directly on the slide. Paula Sent from my iPhone > On Apr 15, 2021, at 11:24 AM, Heather Deziel <hdez...@cnscro.com> wrote: > > > Hi Paula, > > I am cutting at -24, but have tried going as warm as -18. I am currently > learning with 30uM sections with the ultimate goal of moving towards 5 or > 10uM. Our lab standard has been collecting into millonigs solution and doing > free floating IHC. We generally have no issue with this technique, but do > lose some of the peripheral damaged tissue near the infarct in our stroke > brains. We're trying to work up painting the samples directly onto slides > and skipping free floating staining to get a better end product. > > My current samples are very old, they were collected into 4%PFA in 2017 and > cryoprotected by freezing in OCT cryomatrix in a little dish floating on LN2 > in 2019. They've been stored at -80 since then. Usually we process the > brains within a few weeks of collecting them so this particular tissue isn't > our ideal situation. We're using old tissue to practice technique, so the > current samples aren't going to be used for any actual analysis. When we > heard about the tape method of collecting we were very curious as the the > opinion other labs have about it. Have you tried it? > > Thanks for the answer! > > Heather > > Heather Deziel, MSc. > Laboratory Technician, CNS|CRO > 550 University Ave, Charlottetown, PE C1A 4P3 > > (a subsidiary of Neurodyn Life Sciences Inc.) > > > >> On 2021-04-15 10:29, Patpxs wrote: >> >> Good Morning Heather, >> >> I have some questions about how you cut frozen brains. >> >> What temperature are you cutting at? >> How thick are your sections? >> How are your samples frozen? Flash freezing, slow freezing, iso-pentane in >> LN2? >> >> Your answers may provide clues to help you get better cryosections. >> >> Paula >> >> Sent from my iPhone >> >>> On Apr 15, 2021, at 5:39 AM, Heather Deziel via Histonet >>> <histonet@lists.utsouthwestern.edu> wrote: >>> >>> Hello Histonet, >>> >>> I'm looking into working up a tape transfer method of collecting >>> cryosections of brain while preserving infarct to be used in IHC. I >>> find that when I try and section heavily damaged regions of the brain >>> the tissue tears and and I lose it. Has anyone got any recommendations >>> about the the Section-lab transfer tape (Kawamoto method), using the >>> circuit plating tape recommended here >>> (https://www.future-science.com/doi/full/10.2144/btn-2018-0021) or the >>> cryojane system from Leica? >>> >>> Thank you, >>> >>> Heather >>> >>> Heather Deziel, MSc. >>> >>> Laboratory Technician, CNS|CRO 550 University Ave, Charlottetown, PE C1A >>> 4P3 >>> >>> (a subsidiary of Neurodyn Life Sciences Inc.) >>> _______________________________________________ >>> 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
