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Human Blood Cells Protocol

Application Note for Leica EM CPD300 - Life Science Research

Introduction:
Species: Human (Homo sapiens)
Critical point drying of human blood with subsequent platinum / palladium coating and SEM analysis

Procedure:
Sample Holder:
Samples were inserted into the 12 mm cover slip holder

Preparation:
Place 12 mm dia cover slip poly-L-lysine coated in a 12-wells cell culture plate. Add 1 ml 0.85% NaCl in each well to submerge each cover slip.
Pipette gently 50 μl blood on each glass cover slip leave for 5 min at 25°C.
Add 200 μl 0.2 M CaCl2 on top of the blood cells to activate the platelets and leave for 10 min.

Fixation and Dehydration:
Add gently 1 ml of 4% Paraformaldehyde, 0.4% Glutaraldehyde in 0.2 M Sodium Cacodylate Buffer, pH 7.2, on top of the blood cells and leave at least for 10 min. at RT.

Distilled Water3x10 min.
1% aqueous OsO4, 4°C16 h
Distilled water3x10 min.
Ethanol series: 30%, 50%, 70%, 80%, 90%, 96%, 100%1x10 min.
Acetone series: 30%, 50%, 100%1x10 min.

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Leica EM CPD300 auto Program:

Coating:
Mount the dried samples on stubs containing carbon adhesives
Platinum / Palladium coating: 6 nm

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