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and Protocols

Edited by

M. A. Nasser Hajibagheri


Methods in Molecular BiologyTMMethods in Molecular BiologyTM




and Protocols

Edited by

M. A. Nasser Hajibagheri

Page 2

Preparation and Staining of Sections 1


From: Methods in Molecular Biology, vol. 117: Electron Microscopy Methods and Protocols
Edited by: N. Hajibagheri © Humana Press Inc., Totowa, NJ


General Preparation of Material
and Staining of Sections

Heather A. Davies

1. Introduction
This chapter is aimed at those who have not previously done any processing

for electron microscopy (EM). It deals with basic preparation of many different
types of mammalian material for ultrastructural examination; for processing of
plant material (see Hall and Hawes, ref. 1). The material to be processed may
be cell suspensions, particulates, monolayer cultures, or tissue derived from or-
gans. The former three must initially be processed differently from the latter.

For EM, the ultrastructure must be preserved as close to the in vivo situation
as possible. This is done by either chemical or cryofixation; the latter will be
dealt with in later chapters. Aldehydes that crosslink proteins are used for
chemical fixation. Glutaraldehyde, a dialdehyde preserves ultrastucture well
but penetrates slower than the monoaldehyde, paraformaldehyde. Glutaralde-
hyde is used alone for small pieces of material, but a mixture of the two alde-
hydes may be used for perfusion fixation or fixation of larger items.

All reagents used for EM processing must be of high purity. Analytical grade
reagents must be used for all solutions, e.g., buffers and stains. Glutaraldehyde
must be EM grade. For higher purity, distilled or vacuum distilled qualities are
available. Secondary fixation is by osmium tetroxide which reacts with unsat-
urated lipids, is electron-dense and thus stains phospholipids of the cell mem-
brane. This step is followed by dehydration through an ascending concentration
series of solvent before embedding in resin. For simplicity, epoxy resin (Epon)
embedding is described in this chapter; other resins are detailed in Glauert (2)
and Chapters 6 and 7.

Ultramicrotomy and staining ultrathin sections are dealt with briefly; for a
detailed account of the procedure and trouble-shooting (3). The ultrathin sec-

Page 138

Quantitative Aspects of Immunogold Labeling 137

endosomal/lysosomal compartment. In pattern 5, 29 gold particles are specific,
27% are on the Golgi stack, 17% in the CGN, 27% in the TGN and 27% in the
endosomal/lysosomal compartment.

The procedure to calculate the relative distribution could seem long and
tedious but when the background is as low as one to two gold particles/µm2, the
situation is much easier and the LD is very close to LDspec. It is usually the
case. However, when the background is higher and one is to compare two
organelles whose surface sections are very different (one very large compared
with the other one), then the background gold particles will affect very differ-
ently the number of gold particles associated with each compartment. Without
estimation of the specific LD, the relative distribution will not be accurate. It
will be biased toward the large compartment.

Table 3
(A) Estimation of the Specific Labeling Density for for Each Organelle,
LD spec(org) = LD(org)–Background (gold particle\µm2

Golgi endo/
Patterns nucl ER mito Stack CGN TGN lyso cyto

1 0 0 0 8.3 0 0 0 0
2 0 0 0 0 0 0 0 0
3 0 0 1.2 0 0 0 0 0
4 0 0 0 19.5 0 0 1.5? 0
5 0 0.5 0.1 10.9 6.5 4.6 8.2 0

(B) Estimation of the Number of Specific Gold Particles for Each
Organelle, Ng,spec(org) = LDspec(org) ××××× S(org), and the Relative Distri-
bution (RD) of Gold Particles over the Different Organelles Relative Dis-
tribution, (RD,%)

Golgi endo/
Patterns nucl ER mito Stack CGN TGN lyso cyto

1 0 0 0 6 0 0 0 0

2 0 0 0 0 0 0 0 0
3 0 0 0 0 0 0 0 0
4 0 0 0 15 0 0 1�2 0

88% 12%
5 0 0 0 8 5 8 8 0

27% 17% 27% 27%

nucl, Nucleus; ER, Endoplasmic Reticulum; mito, Mitochondria; CGN, Cis Golgi Network;
TGN, Trans Golgi Network; endo/lyso, the endosomal/lysosomal compartment; cyto, Cyto-

Page 139

138 Rabouille

4.5. Linear Density

The determination of the specific labeling density (LD) gives an estimate of
the concentration of gold particles per µm2 of section. That is a meaningful
parameter for the soluble (nonmembrane-bound) antigens which are located in
the lumen of the compartment or in the cytoplasm.

For the membrane-bound antigens, the determination of the labeling density
only (but very importantly) helps in the estimation of the relative distribution.
Their linear density (the number of gold particles per length unit of membrane,
in µm) should be estimated. For instance, the calculation of the relative distri-
bution of gold particles in pattern 5 has shown that four organelles are labeled,
the CGN, the Golgi stack, the TGN, and the endosomal/lysosomal compart-
ment. It could be interesting to know where the linear density of gold particles
is the highest.

Again, that requires the establishment of two parameters:

1. The number of specific gold particles, Nspec(org) associated with the membrane
within a distance of 20 nm (which is the length of the antibody and protein A

2. The length of membrane. The Intersection method (13; Fig. 3B) should be
applied. Membranes of interest are drawn and randomly overlaid with a grid.
Each intersection (I) of the grid with the membrane is counted.

The space between each line of the grid is “d,” so it comes

Length L(org) = I × d/mag (in µm)

The linear density is the specific number of gold particles associated with
the membranes of interest divided by the length of membrane.

Linear density = Nspec(org)/L(org) (in gold particles/µm)

Determination of the linear density can be very time-consuming. For intrac-
ellular organelles, high magnification pictures (90–120 K) and a fine grid (0.2–
0.5 cm) should be used. In case of TGN and CGN, which are networks of
membranes, the measurement of length will be underestimated because not all
membranes are visible. Consequently the linear density can be overestimated.
However, for the plasma membrane, it is a perfect technique.

4.6. Presentation of the Results (12)

Given one pattern, there will be variations from profile to profile within
experiments and from experiment to experiment. The problem is to know when
to stop counting and how to express the results. Pattern 4 of Fig. 1 will be used
to illustrate this point.

Page 276

X-Ray Microanalysis Techniques 275

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