Name: histology basics and cell death detection in honeybee tissue
Uploaded: 2022-07-07T14:00:00
Description: Watch this Scientific Journal Video about Histology Basics and Cell Death Detection in Honeybee Tissue at JoVE.com

슬로베니아 연구 기관, 보조금 번호 P4-133의 지원에 감사드립니다.

1. 꿀벌 연구를위한 기본 조직학
<ol><li>꿀벌 조직의 해부 알림: 일벌을 해부하려면 LED 광원이 있는 해부 현미경을 사용하십시오. 가장 유용한 배율은 ~ 20x입니다.<ol><li>조작 및 해부<ol><li>조심스럽게 집게로 일벌을 가져 와서 얼음 (또는 -20 ° C의 냉동실)에 2 분 동안 넣어고정시킵니다 22. 페트리 접시에 꿀벌을 왼쪽에서 오른쪽으로, 오른쪽에서 왼쪽으로 두 번 흉?...

<ol>
	<li>Ruttner, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Naturgeschichte der Honigbienen. , (1992).</li><li>Jordan, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kleine+Bienenkunde.">Kleine Bienenkunde.</a> &#214;sterreichischer Agrarverlag Wien M&#252;nchen. , 41-45 (1964).</li><li>Snodgrass, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Anatomy+of+the+Honey+Bee.">The Anatomy of the Honey Bee.</a> The Hive and the Honey Bee. , 111-113 (1975).</li><li>Snodgrass, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Anatomy of the Honey Bee. , (2004).</li><li>Hrassnigg, N., Crailsheim, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adaptation+of+hypopharyngeal+gland+development+to+the+brood+status+of+honeybee+(Apis+mellifera+L.)+colonies.">Adaptation of hypopharyngeal gland development to the brood status of honeybee (Apis mellifera L.) colonies.</a> Journal of Insect Physiology. 44 (10), 929-939 (1998).</li><li>Smodi&#353; &#352;kerl, M. I., Gregorc, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characteristics+of+hypopharyngeal+glands+in+honeybees+(Apis+mellifera+carnica)+from+a+nurse+colony.">Characteristics of hypopharyngeal glands in honeybees (Apis mellifera carnica) from a nurse colony.</a> Slovenian Veterinary Research. 52 (2), 67-74 (2015).</li><li>Kubo, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Change+in+the+expression+of+hypopharyngeal-gland+proteins+of+the+worker+honeybees+(Apis+mellifera+L.)+with+age+and/or+role.">Change in the expression of hypopharyngeal-gland proteins of the worker honeybees (Apis mellifera L.) with age and/or role.</a> Journal of Biochemistry. 119 (2), 291-295 (1996).</li><li>Sammataro, D., Yoder, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Honey bee colony health: Challenges and sustainable solutions. , 302 (2012).</li><li>Crailsheim, K., Stolberg, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+diet,+age+and+colony+condition+upon+intestinal+proteolytic+activity+and+size+of+the+hypopharyngeal+glands+in+the+honeybee+(Apis+mellifera+L.).">Influence of diet, age and colony condition upon intestinal proteolytic activity and size of the hypopharyngeal glands in the honeybee (Apis mellifera L.).</a> Journal of Insect Physiology. 35 (8), 595-602 (1998).</li><li>Smodi&#353; &#352;kerl, M. I., Gregorc, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heat+shock+proteins+and+cell+death+in+situ+localisation+in+hypopharyngeal+glands+of+honeybee+(Apis+mellifera+carnica)+workers+after+imidacloprid+or+coumaphos+treatment.">Heat shock proteins and cell death in situ localisation in hypopharyngeal glands of honeybee (Apis mellifera carnica) workers after imidacloprid or coumaphos treatment.</a> Apidologie. 41 (1), 73-86 (2010).</li><li>Gregorc, A., Bowen, I. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+histochemical+characterisation+of+cell+death+in+honeybee+larvae+midgut+after+treatment+with+Paenibacillus+larvae,+Amitraz+and+Oxytetracycline.">The histochemical characterisation of cell death in honeybee larvae midgut after treatment with Paenibacillus larvae, Amitraz and Oxytetracycline.</a> Cell Biology International. 24 (5), 319-324 (2000).</li><li>Higes, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apoptosis+in+the+pathogenesis+of+Nosema+ceranae+(Microsporidia:+Nosematidae)+in+honey+bees+(Apis+mellifera).">Apoptosis in the pathogenesis of Nosema ceranae (Microsporidia: Nosematidae) in honey bees (Apis mellifera).</a> Environmental Microbiology Reports. 5 (4), 530-536 (2013).</li><li>Kurze, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infection+dynamics+of+Nosema+ceranae+in+honey+bee+midgut+and+host+cell+apoptosis.">Infection dynamics of Nosema ceranae in honey bee midgut and host cell apoptosis.</a> Journal of Invertebrate Pathology. 154, 1-4 (2018).</li><li>Johnson, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Honey+bee+toxicology.">Honey bee toxicology.</a> Annual Review of Entomology. 60 (1), 415-434 (2005).</li><li>Gashout, H. A., Guzman-Novoa, E., Goodwin, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthetic+and+natural+acaricides+impair+hygienic+and+foraging+behaviors+of+honey+bees.">Synthetic and natural acaricides impair hygienic and foraging behaviors of honey bees.</a> Apidologie. 51 (6), 1155-1165 (2020).</li><li>McMenamin, A. J., Genersch, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Honey+bee+colony+losses+and+associated+viruses.">Honey bee colony losses and associated viruses.</a> Current Opinion in Insect Science. 8, 121-129 (2015).</li><li>Ramsey, S. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Varroa+destructor+feeds+primarily+on+honey+bee+fat+body+tissue+and+not+hemolymph.">Varroa destructor feeds primarily on honey bee fat body tissue and not hemolymph.</a> Proceedings of the National Academy of Sciences USA. 116 (5), 1792-1801 (2019).</li><li>Requier, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Honey+bee+diet+in+intensive+farmland+habitats+reveals+an+unexpectedly+high+flower+richness+and+a+major+role+of+weeds.">Honey bee diet in intensive farmland habitats reveals an unexpectedly high flower richness and a major role of weeds.</a> Ecological Applications. 25 (4), 881-890 (2015).</li><li>Santos, C., Serr&#227;o, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histology+of+the+ileum+in+bees+(Hymenoptera,+Apoidea).">Histology of the ileum in bees (Hymenoptera, Apoidea).</a> Brazilian Journal of Morphological Sciences. 23 (3), 405-413 (2006).</li><li>Suwannapong, G., Saichon, C., Benbow, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histochemical+comparison+of+the+hypopharyngeal+gland+in+Apis+cerana+Fabricius,+1793+workers+and+Apis+mellifera+Linnaeus,+1758+workers.">Histochemical comparison of the hypopharyngeal gland in Apis cerana Fabricius, 1793 workers and Apis mellifera Linnaeus, 1758 workers.</a> Psyche: A Journal of Entomology. , (2010).</li><li>Ceylan, A., Sevin, S., &#214;zgen&#231;, &. #. 2. 1. 4. ;. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histomorphological+and+histochemical+structure+of+the+midgut+and+hindgut+of+the+Caucasian+honey+bee+(Apis+mellifera+caucasia).">Histomorphological and histochemical structure of the midgut and hindgut of the Caucasian honey bee (Apis mellifera caucasia).</a> Turkish Journal of Veterinary and Animal Sciences. 43 (6), 747-753 (2019).</li><li>Human, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Miscellaneous+standard+methods+for+Apis+mellifera+research.">Miscellaneous standard methods for Apis mellifera research.</a> Journal of Apicultural Research. 52 (4), 1-53 (2013).</li><li>Gregorc, A., Smodi&#353; &#352;kerl, M. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toxicological+and+immunohistochemical+testing+of+honeybees+after+oxalic+and+rotenone+treatments.">Toxicological and immunohistochemical testing of honeybees after oxalic and rotenone treatments.</a> Apidologie. 38 (3), 296-305 (2007).</li><li>Carreck, N. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standard+methods+for+Apis+mellifera+anatomy+and+dissection.">Standard methods for Apis mellifera anatomy and dissection.</a> Journal of Apicultural Research. 52 (4), 1-40 (2013).</li><li>Bowen, I. D., Bowen, S. M., Jones, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Mitosis and apoptosis: Matters of Life and Death. , (1998).</li><li>Eisenberg-Lerner, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Life+and+death+partners:+apoptosis,+autophagy+and+the+cross-talk+between+them.">Life and death partners: apoptosis, autophagy and the cross-talk between them.</a> Cell Death and Differentiation. 16 (7), 966-975 (2009).</li><li>Bowen, I. D., Mullarkey, K., Morgan, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Programmed+cell+death+in+the+salivary+gland+of+the+blow+fly+Calliphora+vomitoria.">Programmed cell death in the salivary gland of the blow fly Calliphora vomitoria.</a> Microscopy Research Techniques. 34, 202-207 (1996).</li><li>D'Arcy, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+death:+a+review+of+the+major+forms+of+apoptosis,+necrosis+and+autophagy.">Cell death: a review of the major forms of apoptosis, necrosis and autophagy.</a> Cell Biology International. 43 (6), 582-592 (2019).</li><li>Matylevitch, N. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apoptosis+and+accidental+cell+death+in+cultured+human+keratinocytes+after+thermal+injury.">Apoptosis and accidental cell death in cultured human keratinocytes after thermal injury.</a> American Journal of Pathology. 153 (2), 567-577 (1998).</li><li>Perry, S. W., Epstein, L. G., Gelbard, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+in+situ+detection+of+apoptosis+and+necrosis+in+monolayer+cultures+by+TUNEL+and+trypan+blue+staining.">Simultaneous in situ detection of apoptosis and necrosis in monolayer cultures by TUNEL and trypan blue staining.</a> BioTechniques. 22 (6), 1102-1106 (1997).</li><li>Cuello-Carrion, F. D., Ciocca, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+detection+of+apoptotic+cells+using+a+modified+in+situ+TUNEL+technique.">Improved detection of apoptotic cells using a modified in situ TUNEL technique.</a> Journal of Histochemistry and Cytochemistry. 47 (6), 837-839 (1999).</li><li>Gregorc, A., Bowen, I. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Programmed+cell+death+in+the+honeybee+(Apis+mellifera+L.)+larvae+midgut.">Programmed cell death in the honeybee (Apis mellifera L.) larvae midgut.</a> Cell Biology International. 21 (3), 151-158 (1997).</li><li>Gregorc, A., Poga&#269;nik, A., Bowen, I. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+death+in+honey+bee+(Apis+mellifera)+larvae+treated+with+oxalic+or+formic+acid.">Cell death in honey bee (Apis mellifera) larvae treated with oxalic or formic acid.</a> Apidologie. 35 (5), 453-460 (2004).</li></ol>

살아있는 유기체에서, 세포 사멸은 아폽토시스 또는 괴사(25)로서 정의되고, 자가포식(autophagy)26을 동반할 수 있다. 세포 사멸과 괴사 세포의 차이점은 세포 사멸은 프로그램 된 세포 사멸의 한 형태이며 정상 세포에서 나타나는 반면 괴사는 치명적인 상태 (예 : 사고, 질병)로 인해 발생한다는 것입니다 27,28. 아폽토시?...

꿀벌 (Apis mellifera L.)은 다른 야생 수분 매개자 중에서도 농업 식물의 가장 중요한 수분 매개자입니다. 수천 년에 걸쳐 변화하는 환경은 꿀벌이 형태, 생리학, 행동 및 여러 병원체와 기생충에 대한 내성을 적응시키는 데 영향을 미쳤습니다. 따라서 꿀벌은 전 세계적으로 매우 다양한 종과 아종을 개발했습니다1. 이러한 결과는 꿀벌의 소화관 구조에 유전 적 변이가 있다는 이전 발견과 일치하지만 중추의 변화가 환경 요인 2,3에 기인 한 것임을 시사합니다.
꿀벌의 소화관에는 앞장, 중추 (심실) 및 뒷부4의 세 가지 주요 부분이 있습니다. 심실은 꽃가루와 꿀/꿀의 소화에 필수적인 기관입니다. 뒷다리에서 삼투압 조절은 물과 이온의 흡수를 통해 발생합니다2. 꿀벌 작업자의 하인두 땀샘 (HPG)은 머리에 위치하며 로열 젤리 성분을 합성하고 분비하여 무리, 여왕 및 식민지 구성원에게 먹이를줍니다. 그들의 크기는 나이와 작업에 따라 변하고 적절한 영양 (품질 꽃가루)에 달려 있습니다. 6 일에서 18 일 사이의 간호사는 양육을 수행하며 HPG의 크기는 5,6 증가합니다. 채집 꿀벌에서 HPG는 퇴화되어 꿀7에서 복잡한 당을 단순한 설탕 (α- 글루코시다 아제, 류신 아릴 아미다 제, 인버 타제)으로 전환하는 데 중요한 효소 만 분비합니다.
꿀벌은 여러 생물학적 및 비생물적스트레스 요인8에 노출되며 소화관은 여러 가지 부정적인 자극제의 영향을 받을 수 있습니다. 병원체로부터 유기체를 보호하는 첫 번째 장벽은 병원균으로부터 보호하기 위해 장 점막으로 구성된 중추의 주변 영양 막입니다4. HPG의 발달과 기능은 식단, 연령 및 집락 상태9에 따라 달라지며 살충제, 살비제10 및 병원균11,12,13의 영향을 받습니다. varroa 방제 처리 및 환경의 살충제로 인한 벌통의 살비제 잔류 물은 수렵 꿀벌과 간호사 꿀벌14,15에 영향을 미칩니다. 꿀벌 식민지에 대한 가장 큰 위협은 진드기 Varroa 소멸자로, 식민지 손실에 기여하는 바이러스 벡터16 및 숙주의 지방 신체 (꿀벌의 중요한 중요한 기관)의 소비자로서 결과적으로 개인의 신체와 식민지 기능에 영향을 미칩니다17.
그러나 집약적 인 농지 서식지는 꿀벌에게 단기적인 식량 공급을 제공 할 수 있습니다. 따라서 농업 환경 계획은 농업 경관에서 꿀 꽃의 가용성을 향상시켜야합니다18. 세포 또는 조직 수준, 특히 중추 및 HPG에서 다른 아종 6,19,20,21 또는 이러한 요인의 치명적인 영향을 평가하기 위해 조직 학적 및 면역 조직 화학적 방법은 꿀벌의 조직학 연구에 사용하기에 실용적이고 충분히 정확합니다.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2-Propanol</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ApopTag Peroxidase kit (ApopTag Peroxidase In Situ Apoptosis Detection)</td>
			<td>Sigma-Aldrich</td>
			<td>S7100</td>
			<td>Assay B, https://www.sigmaaldrich.com/SI/en/product/mm/s7100?gclid=CjwKCA 
			jw7vuUBhBUEiwAEdu2pPanI9SE 
			j81ZTl-nLHEoxXAv7ViKwPA_QRx 
			H7fciMRNcYwR7lbPQbhoCqcQQA 
			vD_BwE; Positive controls included in S7101</td>
		</tr>
		<tr>
			<td>Covers</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DeadEnd Colorimetric TUNEL system</td>
			<td>Promega</td>
			<td>G7360</td>
			<td>Assay A, https://worldwide.promega.com/products/cell-health-assays/apoptosis-assays/deadend-colorimetric-tunel-system/?catNum=G7360</td>
		</tr>
		<tr>
			<td>Dissecting microscope&#160; (for bee dissection)</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Distilled water</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Embedding cassette</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>EnVision System alkaline phosphatase kit</td>
			<td>Dako</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Eosin Y Solution</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td>alcoholic</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td></td>
			<td></td>
			<td>95% (or less pure), 90%, 80%</td>
		</tr>
		<tr>
			<td>Faramount mounting medium, aqueous</td>
			<td>Dako</td>
			<td></td>
			<td>mounting medium</td>
		</tr>
		<tr>
			<td>Flattening table</td>
			<td>Leica</td>
			<td>HI1220</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps&#160; (for bee dissection)</td>
			<td>Fine science tools</td>
			<td>11294-00</td>
			<td>Standard #4</td>
		</tr>
		<tr>
			<td>Formalin 10%</td>
			<td></td>
			<td></td>
			<td>Formaldehyde</td>
		</tr>
		<tr>
			<td>Hematoxylin</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HistoChoice Clearing Agent</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td>clearing agent</td>
		</tr>
		<tr>
			<td>Hydrogen peroxidase 3%</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>BioRad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Insect pins&#160; (for bee dissection)</td>
			<td>Entosphinx</td>
			<td>44594</td>
			<td>Insect pins stainless steel &#8211; white, size 2</td>
		</tr>
		<tr>
			<td>ISCDDK, AP (In Situ Cell Death Detecteion Kit, Alkaline Phosphatase)</td>
			<td>Roche</td>
			<td>11684809910</td>
			<td>Assay C, https://www.sigmaaldrich.com/deepweb/assets/sigmaaldrich/product/documents/362/737/11684809910b 
			ul.pdf</td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Lab clock</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Light microscope</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope slides</td>
			<td></td>
			<td></td>
			<td>Box with the slides must be preserved in a plastic wrap to prevent dust</td>
		</tr>
		<tr>
			<td>Microtome</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Modular tissue embedding station</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Na2HPO4</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde 4%</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Paraplast</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pasteur pipettes</td>
			<td></td>
			<td></td>
			<td>1.5 mL; 3 mL</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish&#160; (for bee dissection)</td>
			<td></td>
			<td></td>
			<td>Filled with condensation silicon&#160; (Xantoprene L blue and Universal liquid plus activator)</td>
		</tr>
		<tr>
			<td>Proteinase K</td>
			<td>Merck</td>
			<td>21627</td>
			<td></td>
		</tr>
		<tr>
			<td>Ringers&#39; solution&#160; (for bee dissection)</td>
			<td></td>
			<td></td>
			<td>7.5 g NaCL, 2.38 g Na2HPO4, 2.72 g KH2PO4, 1 L distilled water</td>
		</tr>
		<tr>
			<td>Scissors&#160; (for bee dissection)</td>
			<td>Fine science tools</td>
			<td>1406-09, 14061-09</td>
			<td>Straight and curved, 9 cm</td>
		</tr>
		<tr>
			<td>Universal liquid plus activator&#160; (for bee dissection)</td>
			<td>Kulzer</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Watchmaker&#8217;s forceps (for bee dissection)</td>
			<td>Fine science tools</td>
			<td>91100-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Water bath</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Watercolor brush</td>
			<td></td>
			<td></td>
			<td>2x</td>
		</tr>
		<tr>
			<td>Xantoprene L blue&#160; (for bee dissection)</td>
			<td>Kulzer</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

histology basics and cell death detection in honeybee tissue

꿀벌 (Apis mellifera L.) 벌집 내부 (간호사 및 기타 벌집 꿀벌)와 벌집 외부 (채집자)는 기후 및 날씨 변화, 다양한 살충제, 병원균 및 영양 실조에 노출되며 주로 입을 통해 들어가고 주로 성인 꿀벌의 소화관에 영향을 미칩니다. 꿀벌에 대한 이러한 외부 및 내부 스트레스 요인의 영향을 이해하고 예방하기 위해 유용한 연구 방법 중 하나는 면역 조직 화학적 방법입니다. 조직 학적 분석을 위해 성인 꿀벌의 중추 (심실) 및 하인두 땀샘 (HPG)을 준비하기위한 기본 프로토콜이 설명됩니다. 세포 손상 수준을 평가하고 조직 재생의 자연적인 과정으로서 괴사를 프로그램 된 세포 사멸 (세포 사멸)과 구별하기위한 상세한 방법론이 설명됩니다. 옥살산과 살충제 (살충제 및 살비제)로 성인 꿀벌을 치료하고 심실 및 HPG에서 세포 사멸을 측정 한 결과가 제시됩니다. 방법론의 장단점도 논의됩니다.

중추의 세포 사멸 감지 류블 랴나에있는 슬로베니아 농업 연구소의 실험 양봉장에서 새로 출현 한 일벌 (Apis mellifera carnica)은 3 % 옥살산 (OA)으로 개별적으로 처리되었습니다.23. OA는 Varroa 소멸자 제어를 위해 양봉에 자주 사용됩니다. 처리 후, 일벌 (각 그룹에서 3 명)을 얼음 위에 고정시켰다. 중추를 해부하여 10 % 포르말린으로 고정시켰다. 그런 다음 ?...

Watch this Scientific Journal Video about Histology Basics and Cell Death Detection in Honeybee Tissue at JoVE.com

Histology Basics and Cell Death Detection in Honeybee Tissue

면역 조직 화학적 방법은 꿀벌 연구에서 성인 꿀벌의 중추 및 하인두 땀샘에서 세포 사멸 및 괴사 수준을 감지하고 평가하는 데 유용합니다.

꿀벌 조직의 조직학 기초 및 세포 사멸 검출

Honeybees (Apis mellifera L.) inside the hive (nurse workers and other hive bees) and outside the hive (foragers) are exposed to climate and weather ...

이 프로토콜은 살충제로 처리 된 꿀벌에 대한 많은 생물학적 및 독성 학적 연구에 유용합니다. Varroa 진드기에 대한 치료를 위해 꿀벌 식민지에서 사용되는 살비제의 꿀벌 조직의 세포 반응을 감지하는 데 도움이됩니다. 꿀벌이나 다른 유익한 곤충의 행동과 함께 조직에 미치는 작은 효과를 감지하는 강력한 도구입니다.시작하려면 조심스럽게 집게로 일벌을 가져 와서 섭씨 영하 20 도의 얼음이나 냉동고에 2 분 동안 올려 고정시킵니다. 페트리 접시에 꿀벌을 흉부의 맨 위 뒤쪽 부분을 통해 대각선으로 왼쪽에서 오른쪽으로, 오른쪽에서 왼쪽으로 두 번 고정합니다. 몸을 덮는 불쌍한 곤충 식염수.페트리 접시를 실체 현미경 아래에 놓고 초점을 맞추고 조정합니다. 중추를 해부하려면 꿀벌 몸의 오른쪽 중앙에있는 tergite A5 아래에 가위의 한 지점을 삽입하여 복부부터 시작하십시오. 테르 기트 A2로 자릅니다. 내부 장기가 손상되지 않도록 가위의 안쪽 날을 몸의 측면과 평행하게 유지하십시오.가위를 왼쪽으로 돌려 자릅니다. 복부의 왼쪽 부분을 부드럽게 열고 고정하십시오. 한 손으로 집게를 사용하여 꿀벌 위를 부드럽게 위로 당기고 다른 손에는 가위로 식도 끝에서 자릅니다.복부에서 위와 중추를 당겨 직장을 자릅니다. 곤충 식염수가 든 피펫을 사용하여 대변이나 조직의 일부를 제거하십시오. 하인두 땀샘의 해부를 위해, 앞에서 설명한대로 얼음 위에 일벌을 고정시킵니다.머리를 잘라 안테나가 위를 향하도록 작은 판에 놓습니다. 두 개의 핀으로 머리를 고정하는데, 하나는 왼쪽 겹눈을 관통하고 다른 하나는 오른쪽 겹눈을 관통합니다. 핀 안쪽의 첫 번째 겹눈을 가로질러 절단하고 음순으로 계속 이동한 다음 두 번째 겹눈을 가로질러 다른 쪽을 다시 자릅니다.안테나를 잘라냅니다. 마스크를 벗고 아직 부착된 부분을 자릅니다. 집게를 가지고 뇌와 겉눈의 일부와 함께 땀샘을 조심스럽게 제거하십시오.헤마톡실린 및 에오신 염색을 위해, 탈랍되고 재수화된 절편을 헤마톡실린에 5분 동안 넣는다. 그런 다음 흐르는 수돗물에 2 분 동안 조심스럽게 두십시오. 그런 다음 증류수에 1 분, 에오신 4 분 동안 넣는다.슬라이드를 96 % 에탄올을 1 분 동안 놓은 다음 2- 프로판올을 2 분 동안 넣고 마지막으로 2 분 동안 클리어링 에이전트에 넣습니다. 장착 매체와 커버 유리를 추가하고 건조시킵니다. 광학 현미경으로 관찰하십시오.코플린 항아리를 준비하십시오. 프로테이나제-K를 준비합니다. 섹션을 탈랍하고 재수화한 후 슬라이드를 PBS에 5분 동안 놓습니다.슬라이드를 용기에 평평하게 놓고 프로테이나제-K를 추가합니다. 증류수로 슬라이드를 씻으십시오. 실온에서 내인성 과산화 효소로 담금질하십시오.슬라이드를 PBS 또는 물로 헹굽니다. 슬라이드를 용기에 평평하게 놓고 실온에서 10초 동안 평형 버퍼를 적용합니다. 조직 주위를 닦은 후 말단 데옥시뉴클레오티딜트랜스퍼라제 효소를 각 섹션에 추가하고 섭씨 37도의 가습 챔버에서 1시간 동안 배양합니다.부화하기 전에 슬라이드 주위의 트레이 안에 축축한 종이 타월을 넣고 플라스틱 랩으로 덮으십시오. 배양 후 표본을 랙에 놓고 세척 중지 버퍼에 두십시오. PBS로 슬라이드를 세척하고 조직 주위를 닦은 후 따뜻한 항 디곡시제닌 퍼 옥시 다제 접합체 2 방울을 섹션에 넣고 가습 용기에서 30 분 동안 배양합니다.PBS로 세척 한 후 작동 강도-퍼 옥시 다제 기질을 준비하고 과산화 효소 기질로 섹션을 덮고 5 분 동안 염색합니다. 슬라이드를 현미경 아래에 놓고 최적의 염색 시간을 결정합니다. 슬라이드를 염색 랙 및 증류수로 세척한 후, 슬라이드를 증류수에서 5분 동안 배양한다.2 분 동안 헤 마톡실린을 사용하여 카운터 염색. 슬라이드를 흐르는 수돗물에 3 분 동안 넣은 다음 증류수에 넣어 씻으십시오. 슬라이드를 유리 커버 슬립과 장착 매체 아래에 장착하고 평평하게 두어 건조시킵니다.광학 현미경으로 관찰하십시오. 영향을 받은 세포의 백분율은 광학 현미경을 사용하여 계산되었습니다. 결과는 옥살산 치료가 중추의 세포에 상당한 영향을 미친다는 것을 나타 냈습니다.면역 염색은 하인두 땀샘의 세포 사멸 핵에서 양성 적색 또는 갈색 반응 생성물을 보였다. 이미다클로프리드 또는 쿠마포스 처리 후의 양성 반응 생성물은 대부분의 선 세포에서 결정되었다. 꿀벌 머리의 마스크를 들어 올릴 때 하 인두 땀샘의 일부를 손상 시키거나 잡아 당기지 않도록주의하십시오.이 기술은 꿀벌의 무증상 변화를 감지하고 꿀벌 및 다른 생물체에 대한 다른 환경 영향을 감지하는 데에도 적합합니다.

histology-basics-and-cell-death-detection-in-honeybee-tissue

Research

JoVE Journal

Biology

Actin Co-Sedimentation Assay; for the Analysis of Protein Binding to F-Actin

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates tension and helps maintains cellular shape.  Although the actin cytoskeleton is a rigid structure, it is a dynamic structure that is constantly remodeling.  A number of proteins can bind to the actin cytoskeleton.  The binding of a particular protein to F-actin is often desired to support cell biological observations or to further understand dynamic processes due to remodeling of the actin cytoskeleton. The actin co-sedimentation assay is an in vitro assay routinely used to analyze the binding of specific proteins or protein domains with F-actin.  The basic principles of the assay involve an incubation of the protein of interest (full length or domain of) with F-actin, ultracentrifugation step to pellet F-actin and analysis of the protein co-sedimenting with F-actin.  Actin co-sedimentation assays can be designed accordingly to measure actin binding affinities and in competition assays.

Proteins bind to filamentous actin (F-actin) through distinct actin binding modules. In this video we demonstrate the procedure of actin co-sedimentation, which is an in vitro assay routinely used to analyze proteins or specific domains that bind F-actin.

굴지 공동 침강 분석, F - 굴지에 바인딩 단백질의 분석

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates ...

연구

생물학

Undecalcified Bone Preparation for Histology, Histomorphometry and Fluorochrome Analysis

Undecalcified bone histology demonstrates the micro-architecture of bone. It shows both the mineralised and cellular components of bone, which provides vital information on bone turnover or bone formation and resorption. This has tremendous importance in a variety of clinical and research applications. It yields beautiful images1 and allows for techniques such as fluorochrome assessment and histomorphometry2. Fluorochrome analysis is a technique where fluorescent dyes that bind to calcium are injected at a particular time point, which allows for quantification of the amount of mineralisation at that given time. Histomorphometry is a process of bone quantification at the microscopic level. 
Performing undecalcified bone histology is technically challenging, particularly with large size specimens. It requires variations in technique from those used in standard paraffin embedded histology. This video illustrates the process of producing good quality sections and demonstrates the technical difficulties and methods with which to overcome them. Specimen preparation, fixation and processing are achieved with a manner similar to other soft tissues, however due to the density and lower permeability of bone considerably longer fixation and processing times are required, often taking several weeks. Embedding is achieved using a supporting medium with similar or equal hardness and density to the bone such as methacrylate- based resins, but unlike paraffin infiltration and embedding, this is an irreversible step. Sectioning can be achieved by grinding which produces a thicker section, which is optimal for studies such as fluorochrome analysis. This is best achieved using a diamond blade on a macrotome. Alternatively, thinner sections can be produced for light microscopy and this is achieved using a sledge microtome with a very sharp blade. The sledge microtome provides the additional strength and stability required for large, hard blocks. Resin embedded sections can be stained with a variety of stains, which are demonstrated.

Undecalcified bone histology provides important information for a variety of clinical and research applications. It is technically challenging, particularly with large size specimens. This video illustrates the process of producing good quality sections and demonstrates the technical difficulties and methods with which to overcome them.

조직학, 조직 형태 측정 및 분석을위한 Fluorochrome Undecalcified 본 준비

Undecalcified bone histology demonstrates the micro-architecture of bone. It shows both the mineralised and cellular components of bone, which provides ...

A &#946;-glucuronidase (GUS) Based Cell Death Assay

We have developed a novel transient plant expression system that simultaneously expresses the reporter gene, &beta;-glucuronidase (GUS), with putative positive or negative regulators of cell death. In this system, N. benthamiana leaves are co-infiltrated with a 35S driven expression cassette containing the gene to be analyzed, and the GUS vector pCAMBIA 2301 using Agrobacterium strain LBA4404 as a vehicle. Because live cells are required for GUS expression to occur, loss of GUS activity is expected when this marker gene is co-expressed with positive regulators of cell death. Equally, increased GUS activity is observed when anti-apoptotic genes are used compared to the vector control. As shown below, we have successfully used this system in our lab to analyze both pro- and anti-death players. These include the plant anti-apoptotic Bcl-2 Associated athanoGene (BAG) family, as well as, known mammalian inducers of cell death, such as BAX. Additionally, we have used this system to analyze the death function of specific truncations within proteins, which could provide clues on the possible post-translational modification/activation of these proteins. Here, we present a rapid and sensitive plant based method, as an initial step in investigating the death function of specific genes.

A &amp;#946;-glucuronidase GUS Based Cell Death Assay

Programmed cell death assays commonly used in mammalian systems such as DNA laddering or TUNEL assays, are often difficult to reproduce in plants. In combination with a GUS reporter system, we propose a rapid, plant based transient assay to analyze the potential death properties of specific genes.

β - glucuronidase (거스)을 기반으로 세포를 파괴 분석

We have developed a novel transient plant expression system that simultaneously expresses the reporter gene, &beta;-glucuronidase (GUS), with putative ...

Use of LysoTracker to Detect Programmed Cell Death in Embryos and Differentiating Embryonic Stem Cells

Programmed cell death (PCD) occurs in adults to maintain normal tissue homeostasis and during embryological development to shape tissues and organs1,2,6,7. During development, toxic chemicals or genetic alterations can cause an increase in PCD or change PCD patterns resulting in developmental abnormalities and birth defects3-5. To understand the etiology of these defects, the study of embryos can be complemented with in vitro assays that use differentiating embryonic stem (ES) cells.
Apoptosis is a well-studied form of PCD that involves both intrinsic and extrinsic signaling to activate the caspase enzyme cascade. Characteristic cell changes include membrane blebbing, nuclear shrinking, and DNA fragmentation. Other forms of PCD do not involve caspase activation and may be the end-result of prolonged autophagy. Regardless of the PCD pathway, dying cells need to be removed. In adults, the immune cells perform this function, while in embryos, where the immune system has not yet developed, removal occurs by an alternative mechanism. This mechanism involves neighboring cells (called "non-professional phagocytes") taking on a phagocytic role-they recognize the 'eat me' signal on the surface of the dying cell and engulf it8-10. After engulfment, the debris is brought to the lysosome for degradation. Thus regardless of PCD mechanism, an increase in lysosomal activity can be correlated with increased cell death.
To study PCD, a simple assay to visualize lysosomes in thick tissues and multilayer differentiating cultures can be useful. LysoTracker dye is a highly soluble small molecule that is retained in acidic subcellular compartments such as the lysosome11-13. The dye is taken up by diffusion and through the circulation. Since penetration is not a hindrance, visualization of PCD in thick tissues and multi-layer cultures is possible12,13. In contrast, TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) analysis14, is limited to small samples, histological sections, and monolayer cultures because the procedure requires the entry/permeability of a terminal transferase.
In contrast to Aniline blue, which diffuses and is dissolved by solvents, LysoTracker Red DND-99 is fixable, bright, and stable. Staining can be visualized with standard fluorescent or confocal microscopy in whole-mount or section using aqueous or solvent-based mounting media12,13. Here we describe protocols using this dye to look at PCD in normal and sonic hedgehog null mouse embryos. In addition, we demonstrate analysis of PCD in differentiating ES cell cultures and present a simple quantification method. In summary, LysoTracker staining can be a great complement to other methods of detecting PCD.

We present a simple protocol to visualize regions of programmed cell death (PCD) in mouse embryos and differentiating embryonic stem (ES) cell cultures using a highly soluble dye called LysoTracker.

태아의 프로그래밍 셀 죽음을 감지 할 LysoTracker의 이용 및 배아 줄기 세포를 차별화

Programmed cell death (PCD) occurs in adults to maintain normal tissue homeostasis and during embryological development to shape tissues and ...

RNA In situ Hybridization in Whole Mount Embryos and Cell Histology Adapted for Marine Elasmobranchs

Marine elasmobranchs are valued animal models for biomedical and genomic studies as they are the most primitive vertebrates to have adaptive immunity and have unique mechanisms for osmoregulation 1-3. As the most primitive living jawed-vertebrates with paired appendages, elasmobranchs are an evolutionarily important model, especially for studies in evolution and development. Marine elasmobranchs have also been used to study aquatic toxicology and stress physiology in relationship to climate change 4. Thus, development and adaptation of methodologies is needed to facilitate and expand the use of these primitive vertebrates to multiple biological disciplines. Here I present the successful adaptation of RNA whole mount in situ hybridization and histological techniques to study gene expression and cell histology in elasmobranchs.
Monitoring gene expression is a hallmark tool of developmental biologists, and is widely used to investigate developmental processes 5. RNA whole mount in situ hybridization allows for the visualization and localization of specific gene transcripts in tissues of the developing embryo. The expression pattern of a gene's message can provide insight into what developmental processes and cell fate decisions a gene may control. By comparing the expression pattern of a gene at different developmental stages, insight can be gained into how the role of a gene changes during development.
While whole mount in situ's provides a means to localize gene expression to tissue, histological techniques allow for the identification of differentiated cell types and tissues. Histological stains have varied functions. General stains are used to highlight cell morphology, for example hematoxylin and eosin for general staining of nuclei and cytoplasm, respectively. Other stains can highlight specific cell types. For example, the alcian blue stain reported in this paper is a widely used cationic stain to identify mucosaccharides. Staining of the digestive tract with alcian blue can identify the distribution of goblet cells that produce mucosaccharides. Variations in mucosaccharide constituents on short peptides distinguish goblet cells by function within the digestive tract 6. By using RNA whole mount in situ's and histochemical methods concurrently, cell fate decisions can be linked to gene-specific expression.
Although RNA in situ's and histochemistry are widely used by researchers, their adaptation and use in marine elasmobranchs have met limited and varied success. Here I present protocols developed for elasmobranchs and used on a regular basis in my laboratory. Although further modification of the RNA in situ's hybridization method may be needed to adapt to different species, the protocols described here provide a strong starting point for researchers wanting to adapt the use of marine elasmobranchs to their scientific inquiries.

RNA In situ Hybridization in Whole Mount Embryos and Cell Histology Adapted for Marine Elasmobranchs

By combining methods for RNA whole mount in situ hybridization and histology, gene expression can be linked with cell fate decisions in the developing embryo. These methods have been adapted to marine elasmobranchs and facilitate the use of these animals as model organisms for biomedical, toxicology and comparative studies.

RNA<em&gt; 현장에서</em전체 마운트 태아 및 세포 조직학에서&gt; 하이브리드는 해양 Elasmobranchs을위한 적응

Marine  elasmobranchs are valued animal models for biomedical and genomic studies as  they are the most primitive vertebrates to have adaptive immunity ...

Dissection of the Auditory Bulla in Postnatal Mice: Isolation of the Middle Ear Bones and Histological Analysis

In most mammals, auditory ossicles in the middle ear, including the malleus, incus and stapes, are the smallest bones. In mice, a bony structure called the auditory bulla houses the ossicles, whereas the auditory capsule encloses the inner ear, namely the cochlea and semicircular canals. Murine ossicles are essential for hearing and thus of great interest to researchers in the field of otolaryngology, but their metabolism, development, and evolution are highly relevant to other fields. Altered bone metabolism can affect hearing function in adult mice, and various gene-deficient mice show changes in morphogenesis of auditory ossicles in utero. Although murine auditory ossicles are tiny, their manipulation is feasible if one understands their anatomical orientation and 3D structure. Here, we describe how to dissect the auditory bulla and capsule of postnatal mice and then isolate individual ossicles by removing part of the bulla. We also discuss how to embed the bulla and capsule in different orientations to generate paraffin or frozen sections suitable for preparation of longitudinal, horizontal, or frontal sections of the malleus. Finally, we enumerate anatomical differences between mouse and human auditory ossicles. These methods would be useful in analyzing pathological, developmental and evolutionary aspects of auditory ossicles and the middle ear in mice.

We present a protocol to isolate the auditory bulla, capsule, and ossicles from postnatal mice for whole mount and histological analysis.

출생 후의 쥐의 청각 수포의 해부 : 중이의 뼈와 조직 학적 분석의 분리

In most mammals, auditory ossicles in the middle ear, including the malleus, incus and stapes, are the smallest bones. In mice, a bony structure called ...

Proteolytically Degraded Alginate Hydrogels and Hydrophobic Microbioreactors for Porcine Oocyte Encapsulation

In reproductive biology, the biotechnology revolution that began with artificial insemination and embryo transfer technology led to the development of assisted reproduction techniques such as oocyte in vitro maturation (IVM), in vitro fertilization (IVF) and cloning of domestic animals by nuclear transfer from somatic cell. IVM is the method particularly of significance. It is the platform technology for the supply of mature, good quality oocytes for applications such as reduction of the generation interval in commercially important or endangered species, research concerning in vitro human reproduction, and production of transgenic animals for cell therapies. The term oocyte quality includes its competence to complete maturation, be fertilized, thereby resulting in healthy offspring. This means that oocytes of good quality are paramount for successful fertilization including IVF procedures. This poses many difficulties to develop a reliable culture method that would support growth not only of human oocytes but also of other large mammalian species. The first step in IVM is the in vitro culture of oocytes. This work describes two protocols for the 3D culture of porcine oocytes. In the first, 3D model cumulus-oocyte complexes (COCs) are encapsulated in a fibrin-alginate bead interpenetrating network, in which a mixture of fibrin and alginate are gelled simultaneously. In the second one, COCs are suspended in a drop of medium and encapsulated with fluorinated ethylene propylene (FEP; a copolymer of hexafluoropropylene and tetrafluoroethylene) powder particles to form microbioreactors defined as Liquid Marbles (LM). Both 3D systems maintain the gaseous in vitro culture environment. They also maintain COCs 3D organization by preventing their flattening and consequent disruption of gap junctions, thereby preserving the functional relationship between the oocyte, and surrounding follicular cells.

Presented here are two protocols for the encapsulation of porcine oocytes in 3D culture conditions. In the first, cumulus-oocyte complexes (COCs) are encapsulated in fibrin-alginate beads. In the second, they are enclosed with fluorinated ethylene propylene powder particles (microbioreactors). Both systems ensure optimal conditions to maintain their 3D organization.

포르신 오시테 캡슐화를 위한 프로틸분해 된 알기네이트 하이드로겔 및 소수성 미생물 반응기

In reproductive biology, the biotechnology revolution that began with artificial insemination and embryo transfer technology led to the development of ...

Quantitative Proteomics Workflow using Multiple Reaction Monitoring Based Detection of Proteins from Human Brain Tissue

The proteomic analysis of the human brain tissue over the last decade has greatly enhanced our understanding of the brain. However, brain related disorders continue to be a major contributor of deaths around the world, necessitating the need for even greater understanding of their pathobiology. Traditional antibody-based techniques like western blotting or immunohistochemistry suffer from being low-throughput besides being labor-intensive and qualitative or semi-quantitative. Even conventional mass spectrometry-based shotgun approaches fail to provide conclusive evidence to support a certain hypothesis. Targeted proteomics approaches are largely hypothesis driven and differ from the conventional shotgun proteomics approaches that have been long in use. Multiple reaction monitoring is one such targeted approach that requires the use of a special mass spectrometer called the tandem quadrupole mass spectrometer or triple quadrupole mass spectrometer. In the current study, we have systematically highlighted the major steps involved in performing a successful tandem quadrupole mass spectrometry-based proteomics workflow using human brain tissue with an aim to introduce this workflow to a broader research community.

The protocol aims to introduce the use of a triple quadrupole mass spectrometer for Multiple Reaction Monitoring (MRM) of proteins from clinical samples. We have provided a systematic workflow starting from sample preparation to data analysis for clinical samples with all the necessary precautions to be taken.

인간 뇌 조직에서 단백질의 다중 반응 모니터링 기반 검출을 이용한 정량적 프로테오믹스 워크플로우

The proteomic analysis of the human brain tissue over the last decade has greatly enhanced our understanding of the brain. However, brain related ...

지방 조직 유래 줄기세포 분리 및 특성 분석

Isolation and Identification of Mesenchymal Stem Cells Derived from Adipose Tissue of Sprague Dawley Rats

Adult mesenchymal cells have revolutionized molecular and cell biology in recent decades. They can differentiate into different specialized cell types, in addition to their great capacity for self-renewal, migration, and proliferation. Adipose tissue is one of the least invasive and most accessible sources of mesenchymal cells. It has also been reported to have higher yields compared to other sources, as well as superior immunomodulatory properties. Recently, different procedures for obtaining adult mesenchymal cells from different tissue sources and animal species have been published. After evaluating the criteria of some authors, we standardized a methodology that is applicable to different purposes and easily reproducible. A pool of stromal vascular fraction (SVF) from perirenal and epididymal adipose tissue allowed us to develop primary cultures with optimal morphology and functionality. The cells were observed adhered to the plastic surface for 24 h, and exhibited a fibroblast-like morphology, with prolongations and a tendency to form colonies. Flow cytometry (FC) and immunofluorescence (IF) techniques were used to assess the expression of the membrane markers CD105, CD9, CD63, CD31, and CD34. The ability of adipose-derived stem cells (ASCs) to differentiate into the adipogenic lineage was also assessed using a cocktail of factors (4 &#181;M insulin, 0.5 mM 3-methyl-iso-butyl-xanthine, and 1 &#181;M dexamethasone). After 48 h, a gradual loss of fibroblastoid morphology was observed, and at 12 days, the presence of lipid droplets positive to oil red staining was confirmed. In summary, a procedure is proposed to obtain optimal and functional ASC cultures for application in regenerative medicine.

저자 스포트라이트: Sprague Dawley Rats의 지방 조직에서 유래한 중간엽 줄기 세포의 분리 및 식별  

This protocol describes a methodology for isolating and identifying adipose tissue-derived mesenchymal stem cells (MSCs) from Sprague Dawley rats.

Sprague Dawley 쥐의 지방 조직에서 유래한 중간엽 줄기 세포의 분리 및 식별

Adult mesenchymal cells have revolutionized molecular and cell biology in recent decades. They can differentiate into different specialized cell types, in ...