Comparisons of Techniques - Sample Preparation Topic for Transmission Electron Microscopy - page 227

The different techniques are necessary for viewing all of the structures of the
membrane junctions and in order to take their functions into account.
3.4.3 Comparison of “Chemical Fixation,” “Cryo-embedding,”
and “Immunolabeling” Techniques
Chemical method: chemical fixation (“Techniques” Chapter 2, Section 11) and
substitution, infiltration, and embedding in cryogenic mode (“Techniques”
Chapter 2, Section 10).
Ultramicrotomy (“Techniques” Chapter 4, Section 4) and positive-staining
contrast (“Techniques” Chapter 7, Section 3) and Immunolabeling
(“Techniques” Chapter 7, Section 4)
Bulk material: normal human dermis
Comparison discussion : Labeling is performed on ultrathin sections after “pro-
gressive low temperature” (PLT) embedding in Lowicryl K4M. At first, a chemical
method is used, and then a cryo-method is used for the embedding. Hyaluronic
acid is combined with a protein fragment, making it possible to make an antibody
labeled with 10-nm gold particles. Hyaluronic acid is highly present in the dermis;
it is found around the fibroblast, type 1 collagen fibers, and elastin, as shown in
Fig. 8.48. It has an important role in maintaining skin tone, which is why it is fre-
quently used in cosmetology. The advantage of the PLT method is that it can be used
to properly carry out immunolabeling on extracellular proteins which are extracted
by purely chemical methods. PLT method preserves the ultrastructure close to that
of the purely chemical method (Fig. 8.47) .
Fig. 8.47 TEM image of a
portion of dermis with a cell
called a fibroblast and the
extracellular glycoproteins
elastin and collagen prepared
by ultramicrotomy using
classic chemical fixation and
embedding at room
temperature. ( M. Haftek,
Dermatologie UCB-Lyon 1 )