First Department of Surgery, Hyogo College of Medicine, Nishinomiya, 663-8501
Received for publication August 7, 1996 and in revised form April 30, 1998
In a retrospective analysis of 174 formalin fixed, paraffin-embedded, hepatocellular carcinoma tissue samples, the correlation between the coefficient of variation (CV) as determined by flow cytometry and the duration of formalin fixation (DFF) was investigated. In addition a potential adverse effect of prolonged formalin fixation on CV and fluorescence intensity in paraffin-embedded liver tissue was also investigated prospectively in samples from four patients in which the DFF varied from three days to four weeks. In the retrospective study, there was a significant linear correlation between the CV and DFF (p<0.001), with the CV increasing as the DFF became prolonged. The proportion of samples in which the CV was less than 10.0 was significantly greater (p<0.01) in samples in which the DFF was less than two weeks. In the prospective study, a decrease in fluorescence intensity was observed as the DFF increased. Following two weeks of formalin fixation the fluorescence intensity decreased to 38.1% of the intensity observed in fresh samples. The mean CV was greater than 10.0 in the samples in which the DFF exceeded two weeks. It is concluded that prolonged formalin fixation has an adverse effect on CV. The DFF for flow cytometric analysis of paraffin-embedded tissue should be no greater than two weeks.
Adhesion Molecule and Cytokines of Hypertensive Rat Arteries
Keiji Suzuki, Kyoumi Nakazato and Masamitu Takama*
Division of Histopathology, Department of Laboratory Sciences, Gunma University School of Health Sciences, 3-39-15 Showamachi, Maebashi, Gunma 371-8514 and * Geriatrics Research Institute, Maebashi Gunma 371-0847
Received for publication August 19, 1997 and in revised form May 11, 1998
The mesenteric arteries of hypertensive rats with bilaterally constricted renal arteries showed severe arterial lesions with deposition of fibrinoid substance in the intima and severe medial cell injury. Scanning electron microscopic examination of these arteries revealed neutrophil adhesion to endothelial cells, and endothelial cell injury.
Immunohistochemically, ICAM-1, IL-1$\alpha$, IL-6, IL-8 and TNF-$\alpha$ expression by endothelial cells of uninjured arteries in hypertensive rats were found to be markedly upregulated. The endothelial cells of the injured arteries with many neutrophil and monocyte adhesion similarly showed marked expression of ICAM-1, and were strongly positive for IL-1$\alpha$, IL-6, IL-8, and TNF-$\alpha$. The medial smooth muscle cells positive for IL-1$\alpha$, IL-6, IL-8 or TNF-$\alpha$ could not be detected in both control and hypertensive rat arteries.
These results suggest that the hypertension activates endothelial cells to increase the adhesion molecule and cytokine production, and induces the neutrophil adhesion and migration in the arterial wall resulting in the hypertensive arterial lesions. Expression of cytokines such as IL-1$\alpha$, IL-6, IL-8 and TNF-$\alpha$ in medial muscle cells of rat arteries was not induced by hypertension.
The Separation of Two Reduction Products of Nitroblue Tetrazolium (Nitro BT) in Paramecium caudatum Using a Microphotometric Image-Analysis System
Kensuke Chikamori, Tsutomu Araki* and Katsuyuki Sato**
Department of Chemistry, Naruto University of Education, Takashima, Naruto-cho, Naruto-shi, Tokushima 772-8502, *Department of Systems and Human Science, Graduate School of Engineering Science, Osaka University, Toyonaka, Osaka 560-8531 and **Department of Biology, Naruto University of Education, Takashima, Naruto-cho, Naruto-shi, Tokushima 772-8502
Received for publication September 10, 1997 and in revised form April 27, 1998
Monoformazan (MF) and diformazan (DF) are the reduction products of nitroblue tetrazolium (Nitro BT), commonly used for the histochemical detection of dehydrogenase activity. We designed a microphotometric image-analysis system, and used it to measure the separate amounts of MF and DF in a single Paramecium caudatum. We stained this cell in the presence of succinate and 1-methoxy phenazine methosulfate at 30$\circ$C, and took the observed density images (ODI) at 530 nm and 650 nm every 3 min for 15 min. We used the component-separation method to reconstruct the individual absorption images of MF and DF from a pair of ODIs of the single cell at each reaction time. The estimation of MF and DF quantities in the cell are based on their total absorbance at each reaction time, and molar extinction coefficients at 530 nm and 650 nm, respectively. Our study indicates the difference in the spatial distribution and also appreciable kinetic differences in the formation between MF and DF, and provides a comparison between the kinetic data from the separate measurements and that taken at 590 nm, which is near the isobestic wavelength of the two formazans (585 nm).
Retinoblastoma Susceptibility Gene Product Retrieval in Formalin-fixed, Paraffin-embedded Tissue : A Heating Method for Enhancing Immunohistochemical Staining
Yoshiro Tsuji 1,2, Katsuyuki Kusuzaki 1, Yasusuke Hirasawa 1, Massimo Serra 3, Nicola Baldinim 3 and Mario Campanacci 3
1 Department of Orthopaedic Surgery, Kyoto Prefectural University of Medicine, Kyoto 602-0841, 2 Department of Orthopaedic Surgery, Yokaichi National Hospital, Shiga 527-8505 and 3 Department of Research Oncology, Rizzoli Orthopaedic Institute, Bologna, Italy
Received for publication October 20, 1997 and in revised form February 19, 1998
We examined immunohistochemical methods to visualize the retinoblastoma susceptibility gene product (RB) in formalin-fixed, paraffin-embedded sections of the U2-OS and Saos-2 human osteosarcoma cell lines. Immunocytochemical studies showed clear staining with an anti-RB antibody in 88% of the smeared U2-OS cells and in none of the smeared Saos-2 cells. Antigen retrieval methods evaluated included microwave-oven and autoclave pretreatment of paraffin-embedded sections immersed in Dulbecco's phosphate buffered saline (pH 7.4) prior to incubation with the primary antibody. Strong and discrete staining of U2-OS cell nuclei for RB was seen after autoclave treatment, stronger than that seen following microwave-oven heating. Moreover, neither microwave-oven nor autoclave pretreatment produced positive staining in the nuclei of Saos-2 cells. Therefore, tissues previously considered unsuitable for immunohistochemical analysis of the RB can be studied after autoclave treatment.
1 Departments of Obstetrics & Gynecology, 2 Dermatology, 3 Plastic Surgery, Iwate Medical University, 19-1 Uchimaru, Morioka 020-8505
Received for publication January 5, 1998 and in revised form May 11, 1998
Dual color fluorescence in situ hybridization (FISH) analysis was applied to 22 squamous cell carcinomas (SCC) of the skin in order to detect deletion of the p53 tumor suppressor gene in interphase nuclei using a cosmid probe for p53 and a centromeric probe for chromosome 17. Fewer p53 signals than 17 centromeric signals and zero or one cosmid signals were observed in 5.6$\pm$4.9% (mean$\pm$SD) of controls. Based on this, 20% deletion was established as the cutoff line to define deletion of the p53 gene. Twenty of the 22 SCC cases demonstrated p53 gene deletion according to this criterion. The percentage of cells with deletion ranged from 22% to 59% (33.9$\pm$10.9%). The ratio between the copy number of chromosome 17 centromeres and p53 in the predominant population of the tumor nuclei with p53 gene deletion was either 2/1 or 1/1. Moreover, the major subfraction was composed of chromosome 17 disomic cells in all cases. This suggests that either p53 gene deletion of one allele in disomic cells or the loss of an entire chromosome 17 in aneusomic cells is the main mechanism of p53 gene deletion in SCC of the skin. Immunohistochemical p53 expression was frequently associated with the p53 gene deletion detected by FISH.
Key words: Dual color fluorescence in situ hybridization, p53 deletion, Squamous cell carcinoma, Skin
Light Microscopic Radioautographic Study on RNA Synthesis in the Adrenal Glands of Aging Mice
Yan Liang
Department of Anatomy and Cell Biology, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto 390-8621
Received for publication February 13, 1998 and in revised form April 16, 1998
The aging changes of RNA synthesis and morphology in the adrenal glands of mice from fetal day 19 to 12 months after birth were studied by light microscopic radioautography after 3H-uridine incorporation.
Eight groups of mice, each consisting of a litter of 3 individuals, were used. One hour after intraperitoneal injections of 3H-uridine, the adrenal gland tissues were fixed, embedded, sectioned and radioautographed. Quantitative analysis was made on the light microscopic radioautographs.
The radioautograms revealed that all types of adrenal gland cells were labeled. In each cell, the grains were localized over the nucleus and the cytoplasm and they were more dense in the nucleus than in the cytoplasm. The activity of RNA synthesis of adrenal glands, as expressed by grain counting, was the highest in the cortex and medulla at fetal day 19, then gradually decreased with aging. In the cortex, the number of silver grains was higher in the zona glomerulosa than those in the other cortical zones from fetal day 19 to 12 months after birth. The number of silver grains was higher in the medulla at embryonic stage than those at the postnatal stages.
From these results, it was concluded that the aging changes of RNA synthesis in the adrenal glands of mice were demonstrated.
Localization of Connexin 26, 32 and 43 in Rat Parotid Glands
Chan Young Lee 1, Takashi Muramatsu 1,2 and Masaki Shimono 1,2
1 Department of Pathology, 2 Oral Health Science Center, Tokyo Dental College, 1-2-2, Masago, Mihama-ku, Chiba, 261-8502
Received for publication March 10, 1998
We investigated the distribution and localization of connexin 26, 32, and 43 in rat parotid glands. Immunofluorescence microscopy revealed the presence of spots reacting for connexin 26 and 32 between endpiece cells. A few spots indicating connexin 43 occasionally were observed in the parotid glands. Connexin 43-positive spots were localized around intercalated ducts. No positive spots for these connexins were detected between ductal cells. Double immunofluorescence microscopy revealed that connexin 26-reactive sites could be observed within the same spots as connexin 32. Double immuno-electron microscopy confirmed that connexin 26 and 32 were co-localized in the same gap junction. These results suggest that connexin 26 and 32 are associated with secretory function and permeability between endpiece cells and that connexin 43 is associated with contraction of the myoepithelial cells that surround intercalated ducts in the rat parotid glands.
Demonstration of Calcium Ion Distribution in Calcifying Cells -X-ray Microanalysis and Electron Spectroscopic Imaging after Fixation with NHA-Containing Fixative and Microwave Irradiation-
1 Department of Cell Biology and Anatomy, Tokyo Medical and Dental University, Bunkyo-ku, Yushima, Tokyo 113-8510, 2 Research Institute for New Drags, Shionogi Co. Ltd., Futaba-cho, Toyonaka, Osaka 561-0825 and 3 Department of Oral Pathology, School of Dentistry, University of Okayama, Shikada-cho, Okayama, 702-8031
Received for publication April 9, 1998
We have seen reporting on the calcium ion distribution in cells and tissues with X-ray microanalysis (EDX) since 1978 [9] and with electron energy-loss spectroscopy (ESS) imaging since 1983 [10, 17], under various fixing conditions [12-16, 18-24]. Almost ten years ago, we tried a fixative containing NHA (N,N-naphthaloylhydroxamine) as a chemical precipitant for Ca2+ ions under microwaved conditions [12, 19]. However, the data did not seem to be as sensitive or useful, under conventional electron microscopy and computerized X-ray microanalysis, compared to the two-step replacement method combined with microwave fixation using potassium (K) oxalate followed by potassium (K) antimonate, for the detection of Ca2+ ions [12, 19-21]. Recently, however, we used ESS to analyze same tissue sections which were fixed with NHA-containing fixative with microwave fixation ten years previously. We discovered with ESS analysis that NHA is a sensitive and ideal chemical reagent for calcium ion detection in a cell giving the same view as that of frozen dried or freeze-substituted tissue sections. Calcium was clearly seen and widely distributed in developing chondroblast endoplasmic ultrastructures: ribosomes, cell and endoplasmic membranes, mitochondria, Golgi, and nucleus. However, free mesenchymal cells which were not involved in calcifying functions had only a very small amount of calcium. Calcium was widely distributed in the lacunae of the surrounding space of the chondroblasts and inmature matrix, but the exoplasm of the chondroblasts was almost calcium-free. Other elemental images in the young chondrocytes or osteoblasts, i.e., P$\cdot$L, S$\cdot$L, O$\cdot$K, and N$\cdot$K, were seen clearly superimposed on the cell ultrastructures, i.e., the cell membranes, ribosomes, endoplasmic reticula, mitochondria, Golgi, and nucleus [24].
Key words: Ca distribution in Calcifying cells, Microwaved with NHA fixative, ESS-imaging
Expression of Bone Morphogenetic Proteins (BMPs) in Fractured Mouse Bone Tissue: In Situ Hybridization with Polymerase Chain Reaction (PCR)-derived Antisense DNA Probe
1 Second Department of Pathology, 2 Department of Orthopaedic Surgery, Kobe University School of Medicine, 7-5-1, Kusunoki-cho, Chuo-ku, Kobe 650-0017
Received for publication May 15, 1998 and in revised form June 8, 1998
We investigated the expression of bone morphogenetic protein (BMP)-2, -4 and -6 during fracture healing of mouse tibiae by in situ hybridization (ISH). Twelve-week-old male BALB/c mice were operated on to make a closed fracture on the proximal tibiae. On days 4, 7 and 14 after the operation, the fractured bones were excised, fixed with 4% PFA and decalcified with 20% EDTA to prepare 5-$\mu$m sections. For ISH we employed digoxigenin (DIG) labeled single-stranded DNA probes specific to BMP-2, -4 and -6 generated by uni-directional polymerase chain reaction (PCR) with antisense primer alone. On days 4-14 after fracture, BMP-2 signals were predominantly expressed in proliferating-hypertrophic chondrocytes and in osteoblasts on the surface of the marginal woven bone. Weak expression of BMP-4 and -6 was detected in spindle-shaped mesenchymal cells and at the site of chondrocytic differentiation; it then decreased during the formation of woven bone. These data suggested that BMP-4 and -6 may be important in the early phase; BMP-2 contributed mainly in the mid to later phase of bone fracture repair. BMPs seemed to promote both proliferation and differentiation of mesenchymal cells through the paracrine/autocrine mechanism.
Key words: Bone morphogenetic protein, In situ hybridization, Polymerase chain reaction, Fracture healing
1 Department of Anatomy, Jichi Medical School, 3311 Yakushiji, Minamikawachi-machi, Kawachi-gun, Tochigi 329-0498, 2 Cosmetics Laboratory, Kanebo Ltd., 5-3-28 Kotobuki-cho, Odawara-shi, Kanagawa 250-0002 and 3 Basic Research Laboratory, Kanebo Ltd., 5-3-28 Kotobuki-cho, Odawara-shi, Kanagawa 250-0002
Received for publication May 19, 1998
Although many hair proteins have been investigated biochemically, little information is available on their subcellular distribution in human hair. We report here on the immunoelectron microscopic technique for defining the ultrastructural localization of hair proteins, especially hair keratins, in human scalp hair shafts: a combination of a rapid-freezing, freeze-substitution fixation without chemical fixatives, and subsequent immunocytochemistry (i.e., rapid-freezing immunocytochemistry). The hair shafts were rapid-frozen, freeze-substituted in acetone without chemical fixatives, and then embedded in LR White resin. Subsequently, ultrathin-sectioned samples were stained for hair keratins by an immunogold technique. Rapid-freezing followed by freeze-substitution without chemical fixatives well-preserved not only the fine structure of the hair shafts but also the antigenicity of hair keratins for immunocytochemistry. In the cortex, hair keratins were present mainly on the macrofibrils. In the cuticle, they were also located primarily in the endocuticle, which did not show the fibrous structure like the macrofibrils did. Rapid-freezing immunocytochemistry appears to be the most viable approach for revealing the macromolecular architecture of human hair, which is a completely keratinized tissue and one of the most delicate tissues in preparation for transmission electron microscopy.
Key words: Human scalp hair shaft, Hair keratins, Freeze-substitution fixation, Rapid-freezing immunocytochemistry, Electron microscopy
Department of Oral Pathology, School of Dentistry, Health Sciences University of Hokkaido, 1757 Kanazawa, Ishikari-Tobetsu, Hokkaido 061-0293
Received for publication January 5, 1998 and in revised form on February 20, 1998, March 20, 1998, and May 6, 1998
The purpose of the present study was to investigate the expression pattern of p21/waf-1/cip-1 mRNA on developing and adult oral tissues in rats. The paraffin sections were obtained from the lower and upper jaws of 18-day rat fetuses and of 2-month-old Sprague-Dawley rats. In order to observe the expression pattern of the p21/waf-1/cip-1 mRNA on the tissue sections, the in situ hybridization method was carried out. In the fetal oral tissues, dental papilla next to the internal dental epithelium, and preodonto-blasts expressed the mRNA. In the adult oral tissues, the localization of p21/waf-1/cip-1 mRNA was observed in the suprabasal, spinous and granular layers of the oral epithelium. The late maturation stage of ameloblasts in the incisor, the osteoblasts, and the cementoblasts and the fibroblast-like cells of the acellular cementum-side in the periodontal ligamant also expressed the mRNA. These results indicated that p21/ waf-1/cip-1 mRNA was expressed at the area of the terminal differentiation in the dental tissues.
Key words: p21/waf-1/cip-1, In situ hybridization, Terminal differentiation, Dental tissue, Development