Application Note
Embedding of murine fibroblasts grown on Aclar
plastics for TEM
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related instrument Leica EM TP
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Embedding of murine fibroblasts grown on Aclar
plastics for TEM
TISSUE CULTURE
Epithelial cells (PtK2) or fibroblasts (3T3) were grown in DMEM at 37C and 8% CO2. They were plated either
on glass coverslips, 12 mm in diameter, or on Aclar plastics (EMS, Science Services, Munich, Germany) of the
same size. The substrata were cleaned by HCl treatment for 2 h and subsequent washing in distilled water
and 70% ethanol. Before use, they were incubated in culture medium at 37°C over night.
PREFIXATION
Prefixation was performed outside of the Leica EM TP, either for 30 min with 3% glutaraldehyde in 0.1 M
cacodylate buffer, pH 7.3 at room temperature, or by microwave fixation in 0.5% glutaraldehyde, as described
in detail elsewhere (Reipert et al., 2008).
USE OF THE LEICA EM TP FOR POSTFIXATION, DEHYDRATION AND EMBEDDING IN EPOXY RESIN
Subsequent processing steps for epoxy resin embedding were performed or in an automatic tissue processor
(LEICA Microsystems, Vienna, Austria). Four baskets (large size, 3 divisions) of the tissue processor were
arranged in pairs of two at the stem assembly to give space for the uptake of 6 coverslips in vertical position
with the cells facing towards the stem. If necessary, further 6 coverslips might be added by placing them with
their back sides opposite to the coverslips already loaded.
Postfixation with 0.5% OsO4, washing in cacodylate buffer, dehydration in a series of ethanol, mediation of
the resin infiltration by propylene oxide, as well as sample infiltration in mixtures of propylene oxide and Agar
100 (Agar Scientific, Cambridge, UK) were performed according to the protocol on the right side (page 3).
Subsequently, the coverslips were taken out of the baskets for flat embedding in pure resin Agar 100 (Agar
Scientific, Cambridge, UK). Cells were infiltrated in a drop of resin for 2 h or over night. After infiltration, an
Eppendorf tube with the bottom and lid cut off was placed above the sample. After an initial heat polymeriza-
tion at 60°C for 3 h the Eppendorf tube was filled up with resin and polymerization was continued over night.
Reipert S, Kotisch H, Wysoudil B, Wiche G. (2008) Rapid Microwave Fixation of Cell Monolayers Preserves
Microtubule-associated Cell Structures. J Histochem Cytochem.
Apr 14. [Epub ahead of print]
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LNT Application Note - EMBEDDING OF MURINE FIBROBLASTS
Fig. 1: MW-fixed and TP-embedded 3T3 fibroblasts (overview)
Bar, 50 µm
Fig. 2: MW-fixed and TP-embedded 3T3 fibroblasts (detail)
Bar, 2 µm
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Leica EM TP Application Note Embedding of murine Fibroblasts 10/2014 ∙ Copyright © by
Leica Mikrosysteme GmbH, Vienna, Austria, 2014. Subject to modifications. LEICA and the
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