• Molecules and Cells
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• 제24권1호
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• pp.76-82
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• 2007

By combining in situ hybridization (ISH) and immunocytochemistry (IC), microscopic topological localization of mRNAs and proteins can be determined. Although this technique can be applied to a variety of tissues, it is particularly important for use on neuronal cells which are morphologically complex and in which specific mRNAs and proteins are located in distinct subcellular domains such as dendrites and dendritic spines. One common technical problem for combined ISH and IC is that the signal for immunocytochemical localization of proteins often becomes much weaker after conducting ISH. In this manuscript, we report a simplified but robust protocol that allows immunocytochemical localization of proteins after ISH. In this protocol, we fix cultured cortical or hippocampal neurons with 4% paraformaldehyde (PFA), rinse briefly in PBS, and then further fix the cells with $-20^{\circ}C$ methanol. Our method has several major advantages over previously described ones in that (1) it is simple, as it is just consecutive routine fixation procedures, (2) it does not require any special alteration to the fixation procedures such as changes in salt concentration, and (3) it can be used with antibodies that are compatible with either methanol (MeOH-) or PFA-fixed target proteins. To our best knowledge, we are the first to employ this fixation method for fluorescence ISH + IC.

• Animal cells and systems
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• 제5권3호
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• pp.263-266
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• 2001

Metallothionein (MT) is induced in the regenerating rat liver. We have investigated expression of MT gene by RT PCR as well as specific localization of MT by immunocytochemistry in regenerating rat liver after partial hepatectomy (PH). MT mRNA level started to increase from 1 h and reached the peak at 8 h after PH. The level decreased gradually by 24 h, and became similar to that of control group. In the immunocytochemical study, in all groups treated with primary antibody, immunogold particles indicating the presence of MT were evenly distributed throughout both cytoplasm and nucleus of the rat hepatocytes. Within the nucleus, the gold particles appeared to be intensely localized in the areas of euchromatin and nucleolus. Within the cytoplasm, gold particles did not seem to adhere to mitochondria or Iysosomes, but were freely distributed. However, rough endoplasmic reticulum was the obvious compartment on which the gold particles were localized. Time course of MT immunoreactivity revealed that distribution of gold particles in hepatocytes increased gradually by 24 h, and decreased at 48 h after PH. Briefly, PH resulted in the sharpest increase in the expression of MT mRNA at 8 h and in the immunoreactivity of MT at 24 h, respectively. It is suggested that the increase of MT mRNA expression, the intensity of immunoreactivity and the specific localization of MT may be associated with the compensatory cell proliferation followed by PH.

• Molecules and Cells
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• 제19권3호
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• pp.408-417
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• 2005

Nitric oxide (NO) occurs in various types of cells in the central nervous system. We studied the distribution and morphology of neuronal nitric oxide synthase (NOS)-containing neurons in the visual cortex of mouse and rabbit with antibody immunocytochemistry. We also compared this labeling to that of calbindin D28K, calretinin, and parvalbumin. Staining for NOS was seen both in the specific layers and in selective cell types. The densest concentration of intense anti-NOS immunoreactive (IR) neurons was found in layer VI, while the weak anti-NOS-IR neurons were found in layer II/III in both animals. The NOS-IR neurons varied in morphology. The large majority of NOS-IR neurons were round or oval cells with many dendrites coursing in all directions. Two-color immunofluorescence revealed that only 16.7% of the NOS-IR cells were double-labeled with calbindin D28K in the mouse visual cortex, while more than half (51.7%) of the NOS-IR cells were double-labeled with calretinin and 25.0% of the NOS-IR cells were double-labeled with parvalbumin in mouse. By contrast, 92.4% of the NOS-IR neurons expressed calbindin D28K while only 2.5% of the NOS-IR neurons expressed calretinin in the rabbit visual cortex. In contrast with the mouse, none of the NOS-IR cells in the rabbit visual cortex were double-labeled with parvalbumin. The results indicate that neurons in the visual cortex of both animals express NOS in specific layers and cell types, which do not correlate with the expression of calbindin D28K, calretinin or parvalbumin between the two animals.

• 한국생명과학회:학술대회논문집
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• 한국생명과학회 2007년도 제48회 학술심포지움 및 추계국제학술대회
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• pp.57.1-57.1
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• 2007

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• Journal of Plant Biology
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• 제39권2호
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• pp.123-126
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• 1996

The pattern of seed storage protein, vicilin, deposition and site of intracellular localization was examined in cotyledon cells of pea (Pisum sativum) seed using the immunocytochemical methods. The vicilin was confined to the cisternae fo the rough endoplasmic reticulum and dictyosome as well as protein granules newly formed in rough endoplasmic reticulum. Vacuolar protein deposites and protein bodies were also labelled by gold particles. After small protein bodies were formed in the rough endoplasmic reticulum, they were transported to large protein bodies and then fused together. Electron dense protein granule, elaborated in the dictyosome, appears to be transported from dictyosome to protein body. A few unlabelled protein granules seem to be accumulated in other type of proteins than vicilin.

• Journal of Periodontal and Implant Science
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• 제27권2호
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• pp.291-303
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• 1997

Periodontal ligament cells may have a role in the regulation of hard and soft periodontal tissues, but their specific function has not yet to be determined. To evaluate further their role in periodontal regeneration, they were examined for osteoblast-like behavior. Periodontal ligament cells and gingival fibroblasts were primarily cultured from extracted premolar with non-periodontal diseases. Cells were cultured with DMEM at $37^{\circ}C$, 5% $CO_2$, 100% humidity incubator, and as a measure of cell characterization, it was examined that the morphology, alkaline phosphatase activity, collagen synthesis, and immunocytochemistry for osteonectin, osteocalcin, and collagen type I. Healthy periodontal ligament cells has more osteoblastic-like cell property in alkaline phosphatase activity. and collagen synthesis than gingival fibroblast. Immunocytochemistry localization explained that calcitonin were expressed in periodontal ligament cells only, and osteonectin and type I collagen were produced in both cells simultaneously. This results indicate that the growth characteristics of periodontal ligament cells and gingival fibroblasts exhibit some differences in proliferative rates and biochemical synthesis. The differences may help to calrify the role such cells play in the regenearation of periodontal tissues.

• 한국수정란이식학회:학술대회논문집
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• 한국수정란이식학회 2002년도 국제심포지엄
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• pp.78-78
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• 2002

The role of heat shock proteins in shielding organism from environmental stress is illustrated by the large-scale synthesis of these protein by the organism studied to date. However, recent evidence also suggests an important role for heat shock protein in fertilization and early development of mammalian embryos. Effects of elevated in vitro temperature on in vitro produced bovine embryos were analysed in order to determine its impact on the expression of heat shock protein 70 (HSP70) by control and frozen-thawed after in vitro fertilization (IVF) or nuclear transfer (NT). The objective of this study was to assess the developmental potential in vitro produced embryos with using of the various containers and examined expression and localization of heat shock protein 70 after it's frozen -thawed. For the vitrification, in vitro produced embryos at 2 cell, 8 cell and blastocysts stage after IVF and NT were exposed the ethylene glycol 5.5 M freezing solution (EG 5.5) for 30 sec, loaded on each containers such EM grid, straw and cryo-loop and then immediately plunged into liquid nitrogen. Thawed embryos were serially diluted in sucrose solution, each for 1 min, and cultured in CRI-aa medium. Survival rates of the vitrification production were assessed by re-expanded, hatched blastocysts. There were no differences in the survival rates of IVF using EM grid, cryo-loop. However, survival rates by straw were relatively lower than other containers. Only, nuclear transferred embryos survived by using cryo-loop. After IVF or NT, in vitro matured bovine embryos 2 cell, 8 cell and blastocysts subjected to control and thawed conditions were analysed by semiquantitive reverse transcription polymerase chain reaction methods for hsp 70 mRNA expression. Results revealed the expression of hsp 70 mRNA were higher thawed embryos than control embryos. Immunocytochemistry used to localization the hsp70 protein in embryos. Two, 8-cell embryos derived under control condition was evenly distributed in the cytoplasm but appeared as aggregates in some embryos exposed frozen-thawed. However, under control condition, blastocysts displayed aggregate signal while Hsp70 in frozen-thawed blastocysts appeared to be more uniform in distribution.

• 대한수의학회지
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• 제49권4호
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• pp.273-278
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• 2009

Neurofascin, one of the members of L1CAM, has been known to have some important roles during the development of nerve fibers. In order to investigate the role of neurofascin associated with the formation of Schmidt-Lantermann incisure in the sciatic nerve, the localization of neurofascin was studied with electron microscopy, immuno-fluorescence and immuno-electron microscopy. In the electron microscopy, the first formation of Schmidt-Lantermann incisure was checked at postnatal day 6 and the complete form of incisures traversing the whole myelin sheath began to be observed at postnatal day 8. In the immunofluorescence, neurofascin immunoreactive Schmidt-Lantermann incisures were first checked at postnatal day 6 and dramatically increased with aging by postnatal day 56. In the immunoelectron microscopy, neurofascin immunoreactive gold particles at the incisure forming sites were first observed at postnatal day 6 and the number of gold particles was increased as the animal was getting old by postnatal day 56. According to the present study, neurofascin is likely to have some relationships with Schmidt-Lantermann incisure formation.

• Journal of Plant Biology
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• 제39권2호
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• pp.99-105
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• 1996

Immunocytochemistry utilizes the specificity of the antigen-antibody reaction to localize specific antigens in cells or cellular organelles. Here we report the use of monoclonal antibodies, in conjunction with gold-labeled second antibodies to study the ultrastructural localization and tissue distribution of the Mr 98, 000 anionic peroxidase and other wall antigens. The antibody specific for this wall peroxidase, mWP3, labeled mainly the cell wall area. At the tissue level, the Mr 98, 000 peroxidase is located predominantly in the leaf mesophyll, internal coleoptile and sieve elements, but not in the root, as assayed with these procedures. The coleoptile walls were less heavily stained than the walls of leaf mesophyll cells. At the subcellular level, it is localized mainly in intercellular regions of the cell walls. A similar staining pattern was revealed by mWP19, one of anti-$\beta$ glucosidase antibody, though it looked less heavily stained than one with mWP3. In order to serve as a control wall staining using IgM monoclonal antibodies, mWP18 was used. Most of the label is localized over wall regions of cells of the young leaf mesophyll and coleoptile.

• 생명과학회지
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• 제15권3호
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• pp.344-351
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• 2005

산화질소(NO)와 칼슘 결합 단백질은 중추신경계의 다양한 세포들에서 나타나며, 이들은 각각 중요 신호전달 분자와 칼슘 완충 분자이다. 본 연구는 햄스터의 시각피질에서 뇌산화질소 합성효소 (nNOS)와 parvalbumin을 포함하는 뉴런들의 분포와 이들의 co-localization 양상을 면역세포화학적 기법을 이용하여 알아보았다. 햄스터 시각피질에서 parvalbumin에 대한 면역 반응성을 나타내는 뉴런들의 전체 수는 nNOS에 대한 면역 반응성을 보이는 뉴런들의 수보다 17배나 많았다. 가장 큰 차이는 시각피질 제5충에서 발견되었으며, 이곳에서 parvalbumin-면역 반응성 뉴런이 nNOS-면역 반응성 뉴런들의 수보다 54.7배나 높았다. nNOS-또는 parvalbumin-면역 반응성 뉴런들은 크기와 형태, 분포 방식이 시각피질에서 유사하게 나타났다. 그러나 이색 면역형광 기법은 햄스터 시각피질에서 nNOS와 parvalbumin을 모두 발현하는 뉴런은 없음을 보여주었다. 본 연구의 결과는 nNOS와 칼슘 결합 단백질 사이의 co-localization양상이 종간에 차이가 존재함을 나타내며 또한 시각피질에 있는 nNOS-면역 반응성 뉴런들의 다양성과 이질성뿐만 아니라 동물 다양성 이해의 중요성을 함께 제시한다고 볼 수 있다.