Re: [Histonet] Oocyte (frog) embedding and sectioning
Peter - we have done goldfish oocytes and they sound very similar to what you are working with. We make sure that they are in a cluster / wrapped in lens paper or biopsy paper / placed between two biopsy sponges. We then process with a routine automated processor overnight program that makes kind of a little flat slab of eggs. Embed using Surgipath paraffin infiltration/embedding system. We trim the blocks as normal. We then place them on cold trays kind of letting the condensation from the warm blocks going onto the cold tray moisten them up slightly. Cut them on rotary microtomes at 4 or 5 microns using low profile dura edge blades with standard angle for dispo blades. They will cut somewhat well at that point. Make sure they don't over soak if you are soaking - that will cause them to fall out of the blocks. They will shred and look like they are going to be horrible but actually turn out pretty good. Put them on adhesive slides, air dry them until they are white, place in a
56-60 oven to dry overnight and routine stain with H&E. Hope this helps out - I know they are difficult and I imagine that the frogs are even smaller than the fish!! Good Luck,
Peter Reed wrote:I'm fishing for any insights into embedding and sectioning Xenopus oocytes.
I have two main problems: Oocytes don't always stay in the block, as if they
are not perfused with paraffin; Sections shatter on the blade or on water
bath before mounting to slide. Have attempted several strategies:
pretreatment with proteinase K to remove membrane, fixing in MEMFA 1h
followed by PBS rinses and PBS storage, fixing in MEMFA overnight then
proceeding directly to dehydration and embedding, using Paraplast and
PolyFin, changing blade angle between 2.5 and 10 degrees.
Stem Cell Institute
University of Minnesota
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Amy S.Porter, HT(ASCP), QIHC
Michigan State University - Department of Physiology
Division of Human Pathology
Investigative Histopathology Laboratory
2201 Biomedical Physical Sciences Room 2133
East Lansing, MI 48824-3320
Ph: 517-355-6475 ext 1480/1481
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