High-Pressure Freezing and Freeze Substitution of Hep-2 Cells Infected with Chlamydia pneumoniae

Application Note for Leica EM HPM100 - Life Science Research

December 20, 2016

Hep-2 cells infected with Chlamydia pneumoniae were cultured on carbon coated 6 mm Sapphire discs.

Cells were high-pressure frozen in an Leica EM HPM100 using the 6 mm CLEM middle plate with following setup: Sapphire disc with cells, spacer 200 μm, bare Sapphire disc, 2 spacers 200 μm. Ethanol was used as a synchronization fluid to transfer pressure at room temperature prior to cooling.

After freezing, the Sapphire disc was removed from the middle plate in anhydrous acetone at -90°C and immediately transferred into a 2 ml Eppendorf tube containing 2% OsO4 in anhydrous acetone, precooled to -90 °C in an Leica EM AFS2 freeze-substitution unit. Samples were substituted for 8 h at -90°C, 8 h at -60°C, 8 h at -30°C, and 1 h at 0°C with periodic temperature transition gradients of 30°C/h.

Samples were then washed twice with anhydrous acetone at 4°C, immersed in 33% Epon/Araldite in anhydrous acetone at 4°C overnight, followed by 66% Epon/Araldite in anhydrous acetone at 4°C for 6 h, and finally in 100% Epon/Araldite at room temperature for 2 h prior to polymerization at 60°C for at least 24 h in a 1.5 ml Eppendorf tube. Sections were post-stained with uranyl acetate and lead citrate.

NOTE: The use of ethanol as a synchronization fluid during high-pressure freezing is not necessary for cell culture monolayers on Sapphire discs.

A simplified sandwich configuration can be used to provide similar results by placing the Sapphire disc in a CLEM middle plate with cells facing up covered with an aluminium specimen carrier dipped in 1-hexadecene with the 100 μm cavity facing the cells.

Fig.1: Thin section of Hep-2 cells infected with Chlamydia pneumoniae.
Fig.2: Higher magnification of inclusion of cell in figure 1. (C)...Chlamydia pneumoniae cells, (G)...Glycogen granules, (Go)...Golgi, (M)...Mitochondrium, (Mt)...Microtubules, (R)...Ribosomes, (V)...Vesicles. Scale bar 500 nm.
Fig.3: Thin section of Hep-2 cells infected with Chlamydia pneumoniae.
Thin section of Hep-2 cells infected with Chlamydia pneumoniae. Higher magnification of selected area in figure 3. (A)…Actin filaments, (MB)…Mulitvesicular body, (Mt)…Microtubules, (N)…Nucleus, (NP)…Nucleopores, (R)…Ribosomes, (Z)…Zonula adhaerens. Scale bar 1 μm.
Fig.5: Thin section of Hep-2 cells infected with Chlamydia pneumoniae. Higher magnification of selected area in figure 4. (Cp)…Clathrin coated pit, (Mt)…Microtubules, (R)…Ribosomes, (Z)…Zonula adhaerens. Scale bar 200 nm.
 

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