Application Note Embedding of murine fibroblasts grown on Aclar

plastics for TEM

MaterialResearch

Life ScienceResearch

MedicalResearch

IndustrialManufacturing

NaturalResources

related instrument Leica EM TP

2

Embedding of murine fibroblasts grown on Aclar plastics for TEM

TISSUE CULTUREEpithelial 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.

PREFIXATIONPrefixation 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 RESINSubsequent 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]

3LNT 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

4

www.leica-microsystems.com

RELATED PRODUCTS

Leica EM TP

The statement by Ernst Leitz in 1907, “With the User, For the User,” describes the fruitful collaboration with end users and driving force of innovation at Leica Microsystems. We have developed five brand val-ues to live up to this tradition: Pioneering, High-end Quality, Team Spir-it, Dedication to Science, and Continuous Improvement. For us, living up to these values means: Living up to Life.

LIFE SCIENCE DIVISION - NANO TECHNOLOGY LNTThe Leica Microsystems Nano Technology Division’s focus is to provide the most compehensive product portfolio for the preparation of biolog-ical, medical and industrial samples for investigation in the Electron and Light Microscope. Excellent Sample Preparation is a prerequisite for perfect microscopy. Your image starts here!

Leica Microsystems – an international company with a strong network of worldwide customer services:

Active worldwide Tel. Fax

Australia ∙ North Ryde +61 2 8870 3500 2 9878 1055

Austria ∙ Vienna +43 1 486 80 50 0 1 486 80 50 30

Belgium ∙ Diegem +32 2 790 98 50 2 790 98 68

Canada ∙ Concord/Ontario +1 800 248 0123 847 405 0164

Denmark ∙ Ballerup +45 4454 0101 4454 0111

France ∙ Nanterre Cedex +33 811 000 664 1 56 05 23 23

Germany ∙ Wetzlar +49 64 41 29 40 00 64 41 29 41 55

Italy ∙ Milan +39 02 574 861 02 574 03392

Japan ∙ Tokyo +81 3 5421 2800 3 5421 2896

Korea ∙ Seoul +82 2 514 65 43 2 514 65 48

Netherlands ∙ Rijswijk +31 70 4132 100 70 4132 109

People’s Rep. of China ∙ Hong Kong +852 2564 6699 2564 4163

∙ Shanghai +86 21 6387 6606 21 6387 6698

Portugal ∙ Lisbon +351 21 388 9112 21 385 4668

Singapore +65 6550 5999 6773 0628

Spain ∙ Barcelona +34 93 494 95 30 93 494 95 32

Sweden ∙ Kista +46 8 625 45 45 8 625 45 10

Switzerland ∙ Heerbrugg +41 71 726 34 34 71 726 34 44

United Kingdom ∙ Milton Keynes +44 800 298 2344 1908 246312

USA ∙ Buffalo Grove/lllinois +1 800 248 0123 847 405 0164

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

Leica Logo are registered trademarks of Leica Microsystems IR GmbH.