• The Korean Journal of Pain
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• v.9 no.2
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• pp.326-335
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• 1996

Background: Though a number of studies have described the distribution of substance P(SP)-like immunoreactivity in the spinal cord, they have been focused on lamina I and II of the dorsal horn and there are little morphological studies on the topographic distribution and ultrastructure of the SP immunoreactive neurons especially in the ventral horn of the spinal cord. this study was conducted to identify distribution pattern of SP immunoreactive neurons and to difine the synaptic organization of their processes in ventral horn of the thoracic cord of the cat by preembbeding immunocytochemical method using SP antiserum. Methods: Five adults cats of either sex were used and deeply anesthetized by intramuscular injection of ketamine. After removal of the spinal cord, samples of thoracic cord were taken and placed in fresh fixative at $4^{\circ}C$ for 2 hours. Transverse sections $50{\mu}m$ thick were processed using the preembbeding immunocytochemical method and incubated consecutively in the specific primary antibody and the 10% normal goat serum, the rabbit anti-substance P antiserum, the biotin-labelled goat anti-rabbit IgG and finally the avidin-biotin-peroxidase complex. The processed tissue sections were throughly washed and stained in the black with 1% uranyl acetate. Section were examined on a electron microscope. Results: 1) SP immunoreactive neurons were observed in the gray matter around central canal. 2) In lamina I and II SP immunoreactivity was observed in both myelinated and unmyelinated nerve fibers, but in ventral horn only in the unmyelinated nerve fibers. 3) SP immunoreactive axon terminals with small round and large dense core vesicles made chemical synapses onto the dendrites of motor neurons in the ventral horn. Conclusion: SP immunoreactive neurons might play an important role in modulation of motor neurons in the ventral horn of the thoracic cord of the cat.

• Journal of Periodontal and Implant Science
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• v.26 no.4
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• pp.1003-1012
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• 1996

The induction of a phenotype with preoperties may have clinical significance in the acceleration of the wound-healing process. Wound contraction involves a specialized cell known as the myofibroblast. The myofibroblasts can be identified by their intense staining of actin bundles with anti-actin antibody. Tissue-specific actin distribution is correlated with the contractile activity of the myofibroblasts and smooth muscle etc. This study was performed to determine the expression of actin filaments in the cytoplasm of cultured human gingival fibroblsts after GaAs laser(BIOSAER, Korea) irradiation. Human gingival fibroblasts were cultured from explants of normal interdental gingival tissue. The third-generation fibroblasts were used for immunohistochemical study. The cultured fibroblasts were exposed $0.53joule/cm^2$(lmW, 7 mimutes) of energy density, and then observed by immunohistochemical method using, rabbit anti0gelsolin, hen smooth muscle polyclonal antibody(Chemicon international inc.), and biotinylated goat anti-rabbit IgG(Vectastain) 24-, 36-, 48-hour after laser irradiation Following results were obtained ; 1. In nonirradiated cultures, round shaped active fibroblasts with abundant cytoplasm and prominet nucleoli were observed. 2. In 24- and 36-hour cultures after laser irradiation, spindle shaped cells with long process were observed. The intensity of stain was seen in cytoplasm of these modified fibroblasts. 3. In 48-hoour cultures after laser irradiation, stained spindle shape cell were not observed. The results suggest that the effect of the galium-arsenide laser treatment on cultured gingival fibroblasts is the rapid development of cytoplasmic actin filaments.

• Journal of Chest Surgery
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• v.32 no.12
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• pp.1078-1086
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• 1999

배경 :폐포내 fibronectin(FN)의 분포와 역할은 많은 연구자에 의하여 연구되어왔다. 흰주에서 폐의 분화시 FN은 태자에서 폐포의 기저막에 주로 분포되고 간엽조직에서도 관찰되면 분화가 진행되면 폐포막의 간질조직에 FN의 함량이 높아진다. 또 FN은 일반적으로 폐포대식세포(alveolar macrophage)에서 분비되고 폐에 질병이 발생하였을 때 다량의 FN이 폐포대식세포에서 분비된다고 보고되어 있다(Schoenberger 등 1984: Ozaki 등 1990; Rom 등 1987 ; Cordier 등 1990) 저자는 흰쥐의 폐포발생이 진행중인 폐포기 후반에서의 폐포막내 정상적인 FN이니 분포의 변화와폐포를 구성하는 큰 폐포세포(type II pneumocyte)에서의 FN의 분비여부를 면역조직염색법과 전자현미경을 이용하여 추적하고자하였다 실험대상 및 방법: 청정동물실에서 사육한 SPF 흰쥐(Sprague-Dawley 계)를 임신시켜 질도말법을 이용하여 태령을 정한 뒤 태아제 17일 및 20일 출생 제 1일, 2일, 3일, 5일, 및 7일의 신생흰쥐를 실험동물로 사용하였으며 대조군의 흰쥐는 체중 200ㅎ의 건강한 수컷을 사용하였다 흰쥐의 폐조직은 면역조직염색을 위해 rabbit anti rat fibronectin polyclonal antibody를 일차항체로 biotinylated goat anti rabbit IgG를 이차항체로 사용하여 폐실질세포내 FN의 분포를 LM으로 관찰하였고 한편 폐포막을 구성하는 세포 중 큰폐포세포가 FN을 분비하는 세포인지를 추적하기 위해 금과립을 첨가한 항체를 사용하여 큰 폐포세포내 FN의 분포를 EM을 이용해서 추적한 결과 다음과 같은 결과를 얻었다 결과 : 제 17일 및 20일 태아시기의 폐에서의 혈관주위에 강한 FN반응이 관찰되었다 출생후 폐포막의 FN의 활성은 출생후 5일 및 7일에 최고주에 달했다. 출생직후 1-2일경에 혈관의 조직내 FN의 활성이 양성을 나타내지만 3일이후 활성이감소되었다. 폐포대식세포내 FN의 활성은 출생후 증가되었다. 폐조직내 소기관지의 FN의 활성은 출생후 완만하게 상승되었다. 큰 폐포세포는 출생 1-3일에 일정량의 FN 반응이 세포질과 미세융모내에 관찰되었다. 결론 : 이상과 같은 결과로 흰쥐의 폐포의 분화과정이 계속되는 출생후 폐에서 FN의 분비는 7일이내에 성숙흰쥐의 폐포내 반응과 비슷한 반응으르 보이며 이때 폐의 실질조직은 분화가 거의 완료되었을 것으로 사료되었고 큰 폐포세포에서도 FN이 분비되는 것으로 결론지울수 있다.

• Applied Microscopy
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• v.32 no.4
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• pp.385-398
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• 2002

The purpose of the present study is to investigate whether stromal cells supporting specific microenvironment for hematopoiesis of bone marrow are affected by toxicants and therapeutic drugs such as antibiotics and anticancer drugs and whether laminin-1 is associated with such effects. SD rats were intraperitoneally injected with 75 mg/kg of cyclophosphamide which is widely used to treat infant's solid tumor, leukemia and myeloma and sacrificed after 3 days, 1 week, 3 weeks or 5 weeks of injection. The bone marrow extracted and paraffin-sectioned was analyzed using immunohistochemical staining. A part of tissues was subjected to electron microscopy following reaction with rabbit anti-laminin antibody, biotinylated goat anti-rabbit IgG conjugated with 12 nm gold particles, and staining with uranyl acetate. 1. The bone marrow tissue at day 3 post injection with cyclophosphamide displayed dilated venous sinus, partial necrotic death, and decreased number of hematopoietic cells. Laminin-1 was intensively stained in the reticular and adipose tissues. 2. Up to 5 weeks post injection, laminin-1 was stained at a low level in the stromal tissue of bone marrow and the number of hematopoietic cell was increased. 3. Deposition of the gold particle which represents laminin-1 expression was observed at the highest level in the stromal cells of bone marrow obtained 3 days after injection, and decreased after 1 to 5 weeks. These results suggest that stromal cells which play a role in supporting microenvironment for bone marrow hematopoiesis augment induction of laminin-1 expression and activation upon administration of cyclophosphamide.

• The Korean Journal of Zoology
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• v.27 no.2
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• pp.103-116
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• 1984

The structural gene of rabbit hemoglobin was cloned into Pst I site of pBR322 in E. coli. The complementary DNA (cDNA) was synthesized from rabbit globin mRNA with avian myeloblastosis viral reverse transcriptase, and then RNA was destroyed at pH 11. The double stranded cDNA was synthesized with both Klenow fragment of E. coli DNA polymerase I and reverse transcriptase and then the hairpin loop was opened with Sl nuclease. Double stranded cDNA was subsequently tailed with dCTP and annealed to dGMP-tailed vector DNA. After transformation and initial screening of appropriate clones by plasmid size, the cloned colonies were identified by in situ colony hybridization using by plasmid size, the cloned colonies were identified by in situ colony hybridization using $[^32P]$-labeled cDNA probes and characterized the inserts with restriction endonucleases. The expression of cloned globin gene was investigated by standard radioimmunoassay using rat anti-rabbit Hb serum as primary antibody and goat antirat IgG serum as secondary antibody. The result suggested that the chimeric proteins (the part of $\\beta$-lactamase from the vector pBR322 and globin from rabbit) were supposedly produced in E. coli and the product had the antigenic determinant of rabbit hemoglobin.

• Korean Journal of Animal Reproduction
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• v.15 no.1
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• pp.67-77
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• 1991

本 試驗은 受精初期段階에서 생쥐卵子 透明帶의 機能을 糾明하고 種特異的인 精子受容體의 存在 및 構造理解를 위한 基礎硏究로서 ICR 생쥐의 透明帶를 免疫原으로 하여 토끼에서 抗透明帶抗體를 生産하고 免疫分析法 및 生物學的 檢査法으로 生産된 抗體가 생쥐 透明帶에 特異性이 있음을 確認한 후 이 抗體가생쥐 未受精卵의 體外受精에 미치는 影響과 또 이 抗體를 생쥐에 受動免疫하였을 때 受精에 미치는 影響을 調査하였던 바 다음과 같은 結果를 얻었다. 1. 處理區로서 抗血淸을, 對照區로서 對照血淸을 卵子培養液에 各各 0.3~10%로 含有한 培養液에서 생쥐의 未受精卵을 2時間 培養한 後 觀察하였을 때 對照區 卵子들의 透明帶가 正常的인 形態를 나타내었던 反面에, 處理區의 卵子들은 모두 透明帶 表面에 뚜렷한 沈澱層을 나타내었다. 이어 對照區와 處理區의 卵子를 0.1% pronase가 含有된 培養液으로 옮긴 후 觀察하였을 때, 對照區 卵子들의 透明帶가 5~7分만에 完全히 融解한 反面에, 處理區에서는 2~3時間까지 殘存하였다. 2. 處理區와 對照區 卵子를 FITC-goat anti-rabbit IgG가 含有된 培養液에서 養液한 후 螢光顯微鏡下에서 觀察하였을 때 對照區에서 對照血淸을 10% 含有한 培養液에서 培養한 일부 卵子들의 透明帶에서 희미한 螢光이 나타났으나 血淸稀釋度가 增加함에 따라 전혀 螢光을 나타내지 않았던 反面에, 處理區에서는 抗血淸이 1 : 1,000으로 含有된 培養液에서 培養한 卵子의 透明帶에서도 강한 螢光이 나타났다. 3. 帶抗體를 抗原으로 하여 間接酵素免疫分析法을 實施하였을 때 抗血淸과 對照血淸이 PBS에 1 : 200까지 稀釋되었을 때의 O.D.價는 별다른 差異를 나타내지 않았으나 血淸의 稀釋度가 增加함에 따라 O.D.價가 有意한 差異를 나타냄으로써 透明帶에 特異性을 갖는 抗體임을 確認할 수 있었다. 4. 抗血淸과 對照血淸이 各各 0.3~10%까지 含有된 培養液으로 생쥐의 未受精卵을 豫備培養한 다음 體外受精을 實施하였을 때의 受精率은 對照區에서 77.7%~87.7%이었던 反面에, 處理區에서는 0~29.8%로서 抗透明帶抗體가 생쥐卵子의 體外受精을 完全히 또는 顯著히 抑制하였다. 그러나 抗血淸과 對照血淸이 各各 2.5~10%까지 含有된 培養液으로 透明帶를 除去한 未受精卵의 體外受精을 하였을 때의 受精率은 處理區와 對照區에서 各各 93.0~98.3% 및 93.3~94.3%로서 受精抑制現象을 나타내지 않았다. 5. 抗血淸과 正常血淸, 그리고 兩 血淸으로부터 protein A-affinity chromatography에 의해 分離한 immunoglubulin G를 各各 생쥐에 受動免疫하였을 때, 對照區로서 對照血淸을 0.1~0.3ml 또는 control IgG를 1~3ml 投與했을 때의 各各의 受精率이 80.4~90.2%, 82.2~94.4%이었던 反面에, 處理區로서 抗血淸을 0.3ml 또는 immune IgG를 3mg 投與한 생쥐에서는 完全한 受精抑制現象이 確認되었다.

• Applied Microscopy
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• v.24 no.2
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• pp.78-92
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• 1994

Lysozyme has been reported to be present in the secretory granules of the Paneth cell, and lysozyme immunoreactivity has been detected by immunogold method in Paneth cells of the intestine of human, mouse and rat. The present study was aimed at clarifying the intracellular distribution and changes of the lysozyme immunoreactivity in rabbit Paneth cell after common bile duct ligation of rabbit, using the electron microscope immunogold technique. Healthy adult rabbits weighing about 2kg body weight were divided into normal and bile duct ligated groups. Common bile duct ligation was performed aseptically under ether anesthesia. Experimental animals were sacrificed on the 1st, the 3rd, the 5th, the 7th and the 14th day after the operation. Mucosal specimens from the intestinal gland of ileum were fixed in 2.5% glutaraldehyde-1.5% paraformaldehyde, followed by 1% osmium tetroxide, embedded in araldite mixture, cut with LKB-V ultratome. Ultrathin sections were placed on parlodion coated nickel grids (200mesh). The section-bearing grids were floated upside down on the added substance in a moist chamber at room temperature except for the primary antibody step, which was at $4^{\circ}C$. Sections were etched with a saturated solution of sodium m-periodate for 60min. After etching, sections were pretreated with 0.02M tris buffered saline (TBS), pH 8.4, with 1% bovine serum albumin (BSA, Sigma) for 60min, then treated polyclonal rabbit anti-human lysozyme (Dakipatts) diluted 1 : 50 in TBS with 0.1% BSA for 20hr. Subsequently, grids were incubated 60min in biotinylated goat anti rabbit IgG (Amersham) diluted 1 : 100 in TBS with 0.1% BSA. After this, sections were incubated 60min on streptavidin gold G10 (Amersham) diluted 1 : 50 in TBS with 0.1% BSA. After each step, the grids were briefly rinsed with TBS with 0.1% BSA. After the strepavidin gold step, the sections were jet washed with distilled water. Counterstain of the sections performed by uranyl acetate and lead citrate, and observed with JEM 100 CX II electron microscope. Observed results were as follow; 1. Secretory granules of mouse Paneth cells have a lysozyme immunoreactivity and also eosinophil leucocyte of rabbit applied for the positive-control stain, are well labeld with gold particles. 2. Normal rabbit Paneth cells have a lysozyme immunoreactivity restricted on the secretory granules. 3. Amount lysosomes containing myelin figures in the Paneth cells were significantly increased from 5th day after the common bile duct ligation. 4. Immunoreactivity of Paneth cell secretory granules were more activated on the 3rd day after the common bile duct ligation as compared with those of the normal animal. But the lysozyme immunoreactivity were decreased from the 5th day after the common bile duct ligation. 5. Considering the above finding, lysozyme contained Paneth cell are affected following of common bile duct ligation, whereas lysosomes containing myelin-figure do not exhibit any immunoreactive relationship with those of secretory granules.

• Korean Journal of Food Science and Technology
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• v.24 no.3
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• pp.204-208
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• 1992

This study was carried out to develop a practical enzyme-linked immunosorbent assay(ELISA) for the determination of soy protein in processed meat products as a preliminary study. The titer of antiserum raised in rabbit by injection of SDS-treated whole buffer extract(WBE) from isolates soy protein(ISP) was above 1:10,000 in indirect ELISA. When the SDS concentration was higher than 0.03% the antibody-antigen reaction was inhibited significantly. However, the antibody-antigen reaction inhibition was not observed when the SDS concentration was less than 0.02%. The antibodies used in this experiment also reacted with renatured antigen after removing SDS by dialysis, though not better than with SDS-denatured antigen(immunogen). The calibration curve with $100\;{\mu}g/100\;ml$ of sensitivity was obtained in indirect competitive ELISA.