Einschlussmittel

Begonnen von Anton Berg, Januar 20, 2009, 13:30:33 NACHMITTAGS

Vorheriges Thema - Nächstes Thema

Anton Berg

Guten Tag,

in der Pathologie werden bekanntlich Schnellschnitte gefertigt und dies in Kryotechnik.

Ist im Forum bekannt, wie hier vorgegangen wird? Wie und womit werden die Objekte behandelt, infiltiert, damit sie schnell und tiefgefroren geschnitten werden können? Hier seien 6µ-Schnitte möglich.

Für diesbezügliche Auskünfte bin ich dankbar.

MfG Anton Berg
Mit freundlichen Grüßen,   Anton

Rene

Hallo Herr Berg,

I'm not sure about the 'state of the art' methodology for clinical specimen, but I think the tissues are directly frozen on the stub, if needed with a bit of mountant, TissueTec or similar (PVA stuff). Freezing speed is not very critical, in my experience, for this work in LM diagnostics. Sections are adhered to slides, dried, or fixed in aldehyde vapours or by microwave before staining.

My own experience with the freezing microtome is with plant material, buds, twigs and leafs. Especially leafs are very critical, sections shatter very easily. I preserved material with fomalin, infiltrated with sucrose (15%), embedded in agarose on the stub. Cytoplasmic detail was excellent, also chlorophyll autofluorescence was completely preserved. My sections ranged between 12-15 um thick.

Hope that helps,

René.

Horst Wörmann

Hallo Rene,

vielen Dank, das sind genau die Informationen, die wir gesucht haben. Wir wollen ebenfalls Pflanzenmaterial schneiden.
Dazu noch ein paar Fragen:

Bei welcher Temperatur sollte man schneiden?
Sollte man das Messer auch kühlen?
Wozu muß mit Sucrose infiltriert werden -um Eiskristallbildung zu vermeiden?
Stört Agarose bei einer evtl. Färbung?
Gibt es eine Vorschrift oder Veröffentlichung zu der Methode?

Viele Grüße,
Horst Wörmann
Anton Berg

Rene

Hallo Horst und Anton,

well, this was entirely experimental method building, although I'm sure I just did re-invent the wheel... so you could be lucky and stumble on some tips in the old literature.
Sucrose infiltration (indeed cryoprotectant) is desribed also for preserved animal material, as well as infiltration with the Tissue Tec glue stuff. The latter didn't work for me, plant material like leafs are very brittle and fall apart very easily. The agarose (low melting type, 15% sucrose incorporated) tend to protect the epidermal layers nicely. Still some luck is involved to get a good cut nicely on a slide.

Anyway. Material was properly preserved in phosphate buffered formalin, then cut in small pieces and infiltrated with 15% sucrose for a day or so in the fridge. The small leaf strips were then transferred to a big drop of agarose on a slide and submerged, after cooling in the fridge you can cut out a small piece of agarose with the leaf bit. That I glued with some tissue tec on a stub and simply put that on the cold stage (-30oC) within the cryostat. I used one of those freezers with the microtome incorporated entirely within the freezer space. Temperature was around -15oC, and yes the knife is at the same temperature. Sections curl up hopelessly without a special anti-roll plate on top of the knife, the sections slip under this plastic plate which is placed about half a mm or so above the knife. When a couple of sections are made, the antiroll plate is moved out of the way and the sections can be picked up by touching with a (cold) needle. Easiest way to proceed is then to drop them straight in a drop of 50% glycerol on a slide (do that within the freezer space!). I have also stuck them on dry (polysine coated) slides for immuno work, but the chances of success on an undisturbed section were less. Sturdier material (twigs, buds) can be cut without agarose and give less problems with transfer to slides.

I have not attempted normal staining, but DIC and fluorescence results from those sections were stunning.
I have been working on this a couple of years ago, as a microscopist it was utterly rewarding to get those kind of results. However the technique was at the time already completely outdated by the use of confocal machinery...

Hop that will be of help for you, keep us updated,

Rene.

bbb

Einfrieren mit flüssigem Stickstoff oder Trockeneis.
Umschließen mit Tussuetec (auf Basis von Polyethylenglykol und Ethylenglykol).
Wir schneiden auf 4-5mikrometer.
Grüße



Mikroskope:
Ergaval
Primostar
AxioImager2
Zeiss Technival1

Mikrotome:
Reichert Jung Hn40 Schlittenmikrotom
Microm HM 355s Rotationsmikrotom
Leica VT1000S Vibratom

Es lebe die Mikroskopie!