17 Vibrational Microspectroscopy for Analysis of Atherosclerotic Arteries
511
Both IR and Raman spectroscopic images are label-free microscopy techniques.
however, they should be compared with histological staining. Such a comparison
allows for the better understanding of spectroscopic results. therefore, the following
chapter discusses the most common histological stainings used in the assessment of
atherosclerotic plaque.
17.2.4 Histological Examination and Immunostaining
of Atherosclerotic Plaque
Atherosclerotic plaque is a heterogeneous structure. the differences occur in the
localization and distribution of the cells and extracellular matrix. the intraplaque
heterogeneity requires the use of special preparation and histological methods to
characterize the morphology and composition of the plaque. the paraffin method is
the most used in tissue preparation, however to visualize fats and other lipids frozen
sections are necessary. the most common staining techniques with the examples are
presented in Fig. 17.2.
the hematoxylin and eosin (h&E) staining method is useful to visualize the
morphology and cellular heterogeneity of the plaque. hallmarks of the atherosclerotic plaque that can be specified by h&E staining include the presence of macrophages and foam cells as well as the extent of the inflammatory and red blood cells
(Fig. 17.2a). very useful staining method for visualization of plaque morphology is
also gomori’s one Step trichrome. the trichrome method additionally shows collagen fiber deposits and allows for the determination of myofibroblasts and erythrocytes (Fig. 17.2b). Another staining method, which is dedicated to establishing
collagen fiber distribution, is Picro Sirius Red. this procedure can also be used
for the characterization of muscle cells (Fig. 17.2c). the Resorcin-Fuchsin staining method is used to visualize different fibrous proteins of extracellular matrix of
elastin in atherosclerotic plaque (Fig. 17.2d). By using the PtAh (phosphotungstic acid hematoxylin) method, erythrocytes and fibrin deposition can be detected
(Fig. 17.2e). Accordingly, with the progression of atherosclerotic plaque, calcification and matrix proteoglycans may occur. to detect calcium in atherosclerotic
plaques, the von Kossa staining is considered to be one of the most commonly used
methods (Fig. 17.2f). the Alcian Blue method of staining is recommended for detection of matrix proteoglycans (Fig. 17.2). Lipids are the components that require
frozen tissue preparation. they appear in the foam cells and in the extracellular
matrix forming the lipid core. the oil Red o staining method is routinely used to
establish its presence in the plaque (Fig. 17.2h) [74–75].
( violet) stained with resorcin-fuchsin method. f Calcium deposition ( black) stained with van Koss
method. g matrix proteoglycans ( intensive blue) stained with alcian blue method. h Lipids ( red)
stained with oil Red o method. e Femoral artery of Wistar rat. Fibrin deposition ( light blue) and
erythrocytes ( dark blue) stained with PtAh method
511
Both IR and Raman spectroscopic images are label-free microscopy techniques.
however, they should be compared with histological staining. Such a comparison
allows for the better understanding of spectroscopic results. therefore, the following
chapter discusses the most common histological stainings used in the assessment of
atherosclerotic plaque.
17.2.4 Histological Examination and Immunostaining
of Atherosclerotic Plaque
Atherosclerotic plaque is a heterogeneous structure. the differences occur in the
localization and distribution of the cells and extracellular matrix. the intraplaque
heterogeneity requires the use of special preparation and histological methods to
characterize the morphology and composition of the plaque. the paraffin method is
the most used in tissue preparation, however to visualize fats and other lipids frozen
sections are necessary. the most common staining techniques with the examples are
presented in Fig. 17.2.
the hematoxylin and eosin (h&E) staining method is useful to visualize the
morphology and cellular heterogeneity of the plaque. hallmarks of the atherosclerotic plaque that can be specified by h&E staining include the presence of macrophages and foam cells as well as the extent of the inflammatory and red blood cells
(Fig. 17.2a). very useful staining method for visualization of plaque morphology is
also gomori’s one Step trichrome. the trichrome method additionally shows collagen fiber deposits and allows for the determination of myofibroblasts and erythrocytes (Fig. 17.2b). Another staining method, which is dedicated to establishing
collagen fiber distribution, is Picro Sirius Red. this procedure can also be used
for the characterization of muscle cells (Fig. 17.2c). the Resorcin-Fuchsin staining method is used to visualize different fibrous proteins of extracellular matrix of
elastin in atherosclerotic plaque (Fig. 17.2d). By using the PtAh (phosphotungstic acid hematoxylin) method, erythrocytes and fibrin deposition can be detected
(Fig. 17.2e). Accordingly, with the progression of atherosclerotic plaque, calcification and matrix proteoglycans may occur. to detect calcium in atherosclerotic
plaques, the von Kossa staining is considered to be one of the most commonly used
methods (Fig. 17.2f). the Alcian Blue method of staining is recommended for detection of matrix proteoglycans (Fig. 17.2). Lipids are the components that require
frozen tissue preparation. they appear in the foam cells and in the extracellular
matrix forming the lipid core. the oil Red o staining method is routinely used to
establish its presence in the plaque (Fig. 17.2h) [74–75].
( violet) stained with resorcin-fuchsin method. f Calcium deposition ( black) stained with van Koss
method. g matrix proteoglycans ( intensive blue) stained with alcian blue method. h Lipids ( red)
stained with oil Red o method. e Femoral artery of Wistar rat. Fibrin deposition ( light blue) and
erythrocytes ( dark blue) stained with PtAh method