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CORRELATIVE LIGHT AND ELECTRON MICROSCOPIC
DEMONSTRATION OF GLYCOSAMINOGLYCANS IN
SEMITHIN SECTIONS BY THE PERIODIC
ACID-THIOSEMICARBAZIDE-OSMIUM
TETROXIDE METHOD
Yasukazu NAGATO", Haruyuki IWIMA and Tsuyuka KUSHIDA
"Department of Morphology, Schoo1 of Medicine, Tokai University Department of Anatomy (Director: Prof. Tsuyuka KUSHIDA) Tokyo Women's Medical College(Received June 22, 1992)
The periodic acid-thiosemicarbazide-osmium tetroxide (PATO) method was employed for accurate localization of giycosaminoglycans by both light and electron rnicroscopy. The cellular structures containing the macromolecules of glycosaminoglycans were examined in semithin sections, O.4-O.5 ptm in thickness, embedded in a mixture of
2-hydroxypropyl methacrylate, Quetoi 523 and methyl methacrylate. Such sections
permitted correlation of identical staining sites under light microscopy and electron microscopy at 200 kV. A comparison of the results obtained with both micrographs indicated that the reaction materials showed good staining with osmium black and
specific contrast in electron micrographs.
Staining by this method was almost as effective as that by the periodic acid-Schiff (PAS) method in mucous granules of goblet cells and microvilli in the small intestine, basement membrane and microvilli in the kidney, and glycogen in the liver cells, but it was not equal to the PAS method in staining of the cell membrane.
Introduction
A method for the histochemical localization of glycosaminoglycans was initially developed for light microscopy using paraffin sections, and re-cently improved for electron microscopy with thin sections. The periodic acid-thiosemicarbazide-osmium tetroxide (PATO) method was developed for the histochemical localization of glycosamino-glycans in thin sections, in place of the periodic acid-Schiff (PAS) method for light microscopyi).
Some modifications have since been made by
Seligman et al. <1965)2), Rambourg (1967)3), and Courtoy and Simar (1973)4).
In recent years, various kinds of semithin sec-tions, however, have been introduced in the field of correlative light and electron microscopy,
which is a useful tool for determining specific
reactions in identical sites5}N'}. With this approach,
a modified PAS method was first employed for
accurate correlation of identical stainiRg sites in
sections O.2-O.3 ptm in thickness embedded in a water-miscible methacrylate, such as a mixture of
glycol methacrylate (GMA), Quetol 523 and
methyl methacrylate (MMA), by electron
micro-scopy at an accelerating potential of 100 kV8)-iO).
The present study, however, demonstrated the difficulties in correlation of the cellular compo-nents when sections O.2-O.3 ptm in thickness were
used, and therefore, sections O.4--O.5 ptm in
thick-ness were used for the correlative observations using an electron microscope operating at 200 kVii}. Thus, the structural relationships shown in stained sections O.4-O.5 ptm in thickness were
This report describes the localization of PATO reactive products in semithin tissue sections embedded in a new formulation of water-miscibie methacrylates, consisting of a mixture of
2-hy-droxypropyl methacrylate (HPMA), Quetol 523
and MMA. It shows the results of correlative light and electron microscopy concerning deposits after staining with the PATO method. In addition, the
results obtained by the PATO and the PAS
methods were compared using a pair of adjacent
sectlons.
Materials and Methods
Tissue preparations
Small pieces of small intestine, liver and kidney
from a mouse (C3H/He) were fixed in a mixture of 4% formaldehyde and 1-2% glutaraldehyde buffer-ed with cacodylate for 6 hr'3). After washing with the buffer solution, they were dehydrated in a graded alcohol series and embedded in a mixture
of HPMA, Quetol 523 and MMAii). Sections
O.3-2.0 um in thickness were cut from a trimmed block using an ultramicrotome.
Light microscopy
A pair of adjacent sections, O.5--2.0 ptm in thickness, were cut with glass knives, floated on water and mounted on slide glasses which were then kept in an oven at 600C for 5-10 minutes. They were stained by either the PAS method for methacrylate sectionsiO) or a modification of the PATO staining method (described below).
Correlative microscopy
Sections were cut with glass knives at a
thick-ness of O.3-O.5 ptm. The PATO method was
applied to such floating sections using a platinum
wire loop by the following steps:
1) stainig with 1% aqueous periodic acid solu-tion for 30 minutes
2) washing with distilled water for a few
mmutes
3) stainig with 1% thiosemicarbazide in 25%
acetic acid for 40 minutes
4) washing with distilled water for a few
grid
6) drying with air
7) treatmentwithosmiumtetroxidevaporfor
30 minutes according to the method of Kushidai`}
8) washingwithdistilledwaterfor10minutes
and then drying
After observation with a light microscope, iden-tical sites of the sections were examined at a low magnification with an electron microscope operat-ing at 200 kV. Photo- and electron micrographs of identical sites in the same section were then compared.
Results
Fixatives
A large number of fixatives recommended for the PAS method were investigated for the PATO method described here. Satisfactory results were noted with a mixture of formaldehyde and glu-taraldehyde'3}. Some other fixatives including 10%
formalin, buffered picric acid-formaldehydei5),
glu-taraldehyde, acrolein and Bouin were effective for examinations of tissue sections greater than 1.0 ptm by light microscopy alone.
Comparison of PATO with the PAS method
The staining for the PAS method in the pre-vious GMA sections recommended the oxidation with periodic acid for 5-10 minutes. For the
macromolecules ]demonstrated with the PATO
method, a ionger period of periodic oxidation than 30 minutes was required.
After staining with either PATO or PAS in a pair of adjacent sections, an intense PATO reac-tion was produced by some kinds of intracellular components such as microvilli, basement mem-branes, mucous cell granules and mast cell gran-ules which were assumed to be structures rich in
glycosaminoglycans. Sections stained by the
PATO method also exhibited osmium black in the cell membranes although the reaction products were readily indicated with the PAS method when either higher concentration of periodic acid or longer oxidation times were used.
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譲纐§§ δ擶1響or PAS was employed in a pair of adjacent sections of the small intestine from a mouse (Figs. IA and
IB). The patterns of the deposits, which were produced by glycosaminoglycans, were similar with both methods. However, somewhat less in-tense deposits of osmium black were present in some membrane structures as shown in Fig. IA. The glycogen of liver cells was stained by both methods examined here. A similar staining pat-tern was revealed in a pair of adjacent sections (Figs. 2A and 2B). Enzymatic digestion with saliva removed glycqgen particles from the section.
Correlative microscopy
Observations of the same sections by both light and electron microscopy clearly indicated the cytoplasmic structures which contained the gly-cosaminQglycans. Figs. 3A and 3B are a pair of
photo- and electron micrographs showing the
villus of epithelial cells from the small intestine on
a grid. The cellular structures stained with the
PATO rnethod were clearly demonstrated at
higher magnification although the filamentous surface coat was not identified in the outer layer
specific products (Figs. 4A, 4B and 4C). Examina-tion with electron microscopy revealed that less reactive products were present in the endoplasmic
reticulum of Paneth cells (Fig. 4B and 4C).
In oxidized sections, the PATO technique
stained the glycogen of liver cells in both light and
electron microscopy (Figs. 5A, 5B and 5C). Local-ization of glycogen was observed as black staining
sites which were lost after salivary digestion, and
indicated negative to electrons without periodic oxidation. In higher magnification electron micro-graphs, discrete glycogen masses showed strong contrast, consisting of aggregated particles (Fig.
5C).
0smium black was concentrated in the renal tubules and the glomeruli of the kidney (Fig. 6A). The microvilli were usually electron dense sites,
and the basement membrane appeared as dark
lines (Figs. 6B and 6C). Well-developed basal infoldings were generally recognizable in the tubules. Control sections did not show any con-trast of the basement membrane and microvilli.
Explanation of Figures
Plate 1
Fig. IA and IB A pair of photomicrographs of adjacent sections from the small intestine. A 1.0 pm thick section was
stained by either the PATO (A) or PAS (B) method. The staining pattern in the goblet granules and the microvilli is quite similar, but the PATO method reacts faintly with the nucleus, some cellular components and the cell membrane. (×1,100) Fig. 2A and 2B A pair of photomicrQgraphs of adjacent 1.0 ptm thick sections from the liver stained by the PATO (A) and PAS (B) method, respectively. Glycogen granules is equally well stained by both methods. (×1,OOO)
Plate 2
Fig. 3A, 3B and 3C Correlative photo (A) and electron <B and C) micrographs of the intestinal epithelial layer from a
mouse. A O.5 "m thick section was stained by the PATO method. Electron micrQgraphs illustrate the reactive deposit in the goblet granules (g) and the microvilli (mv). Cytoplasmic organella such as the Golgi apparatus (G), the mitochondria (M) and the cell membrane are generally identified. (3A and 3B: ×1,800, 3C: ×6,OOO)
Fig. 4A, 4B and 4C Photo (A) and electron (B and C) micrographs of the same O.5 "m thick section from the crypt of Lieberkuhn, illustrating Paneth cells at the base of the crypt after staining with PATO. Stained granules of photomicrqgraph (A) correlate with the dense granules (g) of the electron micrqgraphs (B and C). Thick layers of the
endoplasmic reticulum (ER) and the mitochondria (M) are well demonstrated. (4A and 4B: ×2,Ooo, 4C: ×6,OOO)
Plate 3
Fig. 5A, 5B and 5C A pair of photo (A) and electron (B and C) micrQgraphs of a O.5 ptm thick section of the liver stained with PATO. Various sizes of glycogen particles tgly) are clearly displayed. N: nucleus <5A and 5B: ×2,1oo, 5C: ×4,500)
Discussion
Periodic acid is a useful oxidizing agent for many types of glycosaminoglycansi6}. PATO and the related methods have become well established for demonstrating glycosaminoglycans in thin sec-tions2}. The limited reactivity of
thiosemicar-bazide is useful in studying specifically the reac-tive aldehyde produced by periodic acid oxidation. .
In photo- and electron micrographs of different
tissues obtained with the PATO method, the
PATO method gave high quality images in semi-thin sections embedded in a mixture of HPMA,
Quetol 523 and MMA. The best results were
obtained after fixation with a mixture of 4% formaldehyde and 1% glutaraldehyde. Under these conditions, the stained images increase in con-trast and a higher magnification thus allows
observation of the fine structures.
With the PATO method, the presence of gly-cosaminoglycans in granules of goblet cells and Paneth cells from the intestinal epithelium,
microvilli of the small intestine and renal tubules,
and kidney basement membrane was
demon-strated in tissue sections of both micrographs.
Glycogen in liver cells is consider to be positive in
the PATO method, since it is not stained in
controls with salivary digestion and nonoxidized treatments. These findings agree with the results･ of the previous histochemical study with PAS in water-miscible methacrylates sections at a high
accuracylO).
When comparing the results of the PATO and PAS methods, partial differences were present. In particular, examination with the PATO method indicated that some cytoplasmic structures such as the Golgi apparatus, the endoplasmic reticulum and the cell membrane, can also be considered as
positive since in control sections these structures
were not contrasted more than in unstained sec-tions. The results suggest that PATO staining is effective for determining ultrastructures which contain glycosaminoglycans when semithin sec-tions are employed with this method although it is obvious that this method dose not provide
histo-chemical demonstration of the total
glycosamino-glycan content in tissue sections.
Correlative light and electron microscopy is a tool in histochemisty for determining the
struc-tural details of identical cells for the visualization of specific reactions which involve particular
sub-stances. In the case of glycogen, the PAS reaction was first employed in semithin sections'embedded in a mixture of GMA, Quetol 523 and MMA with some modifications8}. The glycosaminoglycans in the goblet granules of the small intestine were also correlated by both micrographs using the
PAS method with the methacrylate
embed-ding9)iO}. Although the low electron scattering property of such embedding medium permits
ob-servation of cytoplasmic detail in sections O.2-O.3
ptm in thickness under an electron microscope with an accerelating potential of 100 kV, it is
difficult to examine preservation of reaction
prod-ucts within the section by both light and electron
.mlcroscopy.
Introduction of HPMA, Quetol 523 and MMA as an embedding medium provides satisfactory con-ditions for staining and observing tissue sections O.5 ptm in thickness under an electron microscope operating at 200 kVii). The present examples suc-ceeded in determining accurate localization of the reaction products in sections stained for glyco-saminoglycans. Correlative light and electron microscopy involves a compromise between loss of
stainability or electron density, and the
preserva-tion of the' ultrastructure. Thus, the method allows accurate localization of
glycosaminogly-cans in identical sites with both light and electron
mlcroscopy.
References
1) Hanker JS, Seaman AR, Weiss LP et al:
philic reagents: New cytochemical principle for light and electron microscopy. Science 146: 1039, 1964
2) Seligman AM, Hanker JS, Wasserkrug H et al:
Histochemical demonstration of some oxidized molecules with thiocarbohydrazide (TCH) or carbazide (TSC) and osmium tetroxide. J Histochem Cytochem 13: 629-639, 1965
tech- chem Cyt㏄hem 15：409−4．12，1967 4）Courtoy R， Simar U：Importance of controls for the demonstration of carbohydrates in electron microscopy with the si互ver methenamine or the thiocarbohydrazide− silver proteinate method． J Microsc 100：199−211，1973 5）Nagato Y， Suzuki T， Kushida T et a1：Hist㏄hemical localization of alkaline phosphatase activity using GMA−Quetol 523．embedding by photopolymerization． Okalimas Folla Anat Jpn 57：29−44，1980 6）Nagato Y， Kushida H：Application of ruthenium red staining to the histochemical demonstration of mast cell in semithin section embedded．in glycol methacrylate， Quetol 523 and methyl methacrylateJEIectron Microsc 34＝139−143，1985 7）Rieder CL，駐owser SS＝Correlative immunofluores− cence and electron microscopy on the same s㏄tion of epon・embedded material． J Hist㏄hem Cyt㏄hem 33： 165−171，1985 8）Nagato Y， Kushida T， Kushida H：Light and elec− tron microscopic observations of glycogen in semi−thin sect董ons employing GMA， Quetol 523 and methyl methacrylate． T6kai J Exp Clin Med 7：127−133，1982 9）Nagato Y， Kushida T， Kusb量da H：Amethod for histochemical localization of glycosaminoglycans in semithin tissue sections embedded in GMA， Quetol 523 and methyl methacrylate．∫Electron Microsc 33： periodate−Schiff reactive glycosaminqglycans in semi− thin sections embedded in GMA−Queto1523−MMA． Ap・ plication of a method for correlative light and electron microscopy of identical sites． Tokai∫Exp CIin Med 10： 37−46，1985 11）Kushida H， Kushida T， Iijima H：An improved method for both light and electron microscopy of iden・ tical sites in semi・thin tissue sections under 200 kV transmission electron microscope， J Electron Microsc 34：438−441，1985 12）Nagato Y， Sekiguc血i M， Kushida T et a1：Correla− tive light and electron microscopic observations on ectopic neurons in the cerebellum of dreher mutant mouse． J Electron Microsc 40二11−18，1991 13）Nagato Y， Ktlsllida T， Kushida H：Fixation of sen｝i・ thin section for combined light and electron mic≠oscopy． Okajimas Fo豆ia Anat Jpn 5S：69−80，1981 14）Kusllida H：Handling of semi−thin sections of GAM Queto1523 embedded tissue foセlight and electron microscopy with special reference to osmium tetroxide vapor staining． J Electron Microsc 27：59−60，1978 15）Zamboni L， Martino CD：Buffered picric acid」for・ maldehyde：Anew rapid fixative for electron micro− scopy． J Cell Biol 35：148A，1967 16＞McManus JFA：Histological and histochemical uses of periodic acid． Stain Technol 23：99408，1948 過ヨウ素酸一チオセミカルバジドーオスミウム酸法による準超薄切片の グリコサミノグリカンの光学および電子顕微鏡における対比観察 ナガ ト
