Transmission Electron Microscopy of Astrocytes

TEM was conducted as previously described (Konzack et al., 2015 (link)). The primary cortical astrocytes were fixed in 1% glutaraldehyde and 4% formaldehyde mixture in 0.1 m phosphate buffer for 10 min. The cells were detached, and fixation was continued for 1 h. After fixation, the cells were centrifuged, immersed in 2% agarose in distilled water, postfixed in 1% osmium tetroxide, dehydrated in acetone, and embedded in Epon LX 112 (Ladd Research Industries). Thin sections were cut with a Leica Ultracut UCT ultramicrotome, stained in uranyl acetate and lead citrate, and examined in a Tecnai G2 Spirit TEM (FEI Europe). Images were captured by using a Quemesa CCD camera (Olympus Soft Imaging Solutions GmbH) and analyzed with a Tecnai G2 Spirit 120 kV TEM with Veleta and Quemesa CCD cameras and a Philips CM100 equipped with CCD camera 23.

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Byts N., Sharma S., Laurila J., Paudel P., Miinalainen I., Ronkainen V.P., Hinttala R., Törnquist K., Koivunen P, & Myllyharju J. (2021). Transmembrane Prolyl 4-Hydroxylase is a Novel Regulator of Calcium Signaling in Astrocytes. eNeuro, 8(1), ENEURO.0253-20.2020.

Publication 2020

Epon LX 112 Ultracut UCT ultramicrotome Quemesa CCD camera CM100

Acetone Agarose Astrocytes Buffer Cells Citrate Cortical Epon Formaldehyde Glutaraldehyde Ladd Lx 112 Osmium tetroxide Phosphate Thin sections Ultramicrotome Uranyl acetate

Corresponding Organization :

Other organizations : University of Oulu, Åbo Akademi University

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Variable analysis

independent variables

Fixation method (1% glutaraldehyde and 4% formaldehyde mixture in 0.1 M phosphate buffer)

dependent variables

Ultrastructural features of primary cortical astrocytes observed using transmission electron microscopy (TEM)

control variables

Phosphate buffer concentration (0.1 M)
Fixation duration (10 min initial, 1 h total)
Centrifugation
Agarose embedding
Osmium tetroxide postfixation
Acetone dehydration
Epon LX 112 embedding
Uranyl acetate and lead citrate staining
Tecnai G2 Spirit TEM (FEI Europe) with Veleta and Quemesa CCD cameras
Philips CM100 TEM with CCD camera

controls

Positive control: Not explicitly mentioned.
Negative control: Not explicitly mentioned.

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