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

我非常感谢斯洛文尼亚研究机构的支持，批准号P4-133。

1. 蜜蜂研究的基础组织学
<ol><li>蜜蜂组织解剖 注意：要解剖工蜂，请使用带LED光源的解剖显微镜。最有用的放大倍率是~20倍。<ol><li>操作和解剖<ol><li>小心地用镊子取一只工蜂，并将其放在冰上（或放入-20°C的冰箱）2分钟以固定22。将蜜蜂斜着固定在培养皿上，从左到右和从右到左两次穿过胸部的最上部。</li>
			<li>倒入昆虫盐水覆盖身体。将培养皿放在显微镜下...

<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>

在生物体中，细胞死亡被定义为细胞凋亡或坏死25，并可伴有自噬26。凋亡细胞和坏死细胞之间的区别在于，细胞凋亡是程序性细胞死亡的一种形式，出现在正常细胞中，而坏死是由于致命条件（例如，事故，疾病）而发生的27，28。可以使用基于TUNEL技术的检测试剂盒检测细胞凋亡（通过在细胞凋亡过程中产生的双链D...

蜜蜂（Apis mellifera L.）是其他野生传粉媒介，也是农业植物最重要的传粉媒介。几千年来，不断变化的环境影响了蜜蜂调整其形态、生理、行为和对几种病原体和寄生虫的耐受性。因此，蜜蜂在全球范围内发展出高度多样化的物种和亚种1。这些结果与先前的发现一致，即蜜蜂的消化道结构存在遗传变异，但也表明中肠的改变是由于环境因素2，3。
蜜蜂的消化道有三个主要部分：前肠、中肠（脑室）和后肠4。脑室是消化花粉和花蜜/蜂蜜的重要器官;在后肠中，渗透控制是通过吸收水和离子2进行的。蜜蜂工人的下咽腺（HPG）位于头部，合成和分泌蜂王浆成分，以喂养育雏，蜂王和蜂群成员。它们的大小随着年龄和任务而变化，并取决于适当的营养（优质花粉）。6至18天的护士进行育雏，HPG的大小增加5，6。在觅食蜂中，HPGs退化并仅分泌对将蜂蜜中的复杂糖转化为简单糖（α-葡萄糖苷酶，亮氨酸芳基酰胺酶，转化酶）很重要的酶7。
蜜蜂暴露于几种生物和非生物应激源8，消化道可能受到几种负面兴奋剂的影响。保护生物体免受病原体侵害的第一道屏障是中肠的周养膜，它由肠粘膜组成，以防止病原体4。HPG 的发育和功能取决于饮食、年龄和菌落条件9，并受到杀虫剂、杀螨剂10 和病原体11、12、13 的影响。由于瓦罗亚控制处理和来自环境的杀虫剂，蜂巢中的杀螨剂残留会影响觅食蜂和护士蜂14，15。对蜜蜂群体的最大威胁是螨虫Varroa破坏者，既是导致蜂群损失的病毒载体16，也是宿主肥胖身体（蜜蜂的重要重要器官）的消费者，因此会影响个体的身体和群体功能17。
然而，集约化的农田栖息地可以为蜜蜂提供短期的食物供应。因此，农业环境计划应提高农业景观中蜜花的供应18.为了评估不同亚种6，19，20，21的形态或这些因素在细胞或组织水平上的亚致死作用，特别是中肠和HPG，组织学和免疫组织化学方法是实用的，并且足够准确，可用于蜜蜂的组织学研究。

<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

蜂巢内（护士和其他蜂巢蜜蜂）和蜂巢外（觅食者）的蜜蜂（Apis mellifera L.）暴露于气候和天气变化，各种杀虫剂，病原体和营养不良，主要通过口腔进入，主要影响成年蜜蜂的消化道。为了了解和防止这种外部和内部压力源对蜜蜂的影响，一种有用的研究方法是免疫组织化学方法。描述了准备成年蜜蜂的中肠（脑室）和下咽腺（HPG）以进行组织学分析的基本方案。描述了一种详细的方法来评估细胞损伤水平，并将坏死与作为组织再生自然过程的程序性细胞死亡（细胞凋亡）区分开来。介绍了用草酸和杀虫剂（杀虫剂和杀螨剂）处理成年蜜蜂的结果以及脑室和HPG中细胞死亡的测定。还讨论了该方法的优缺点。

中肠细胞死亡检测 来自卢布尔雅那斯洛文尼亚农业研究所实验养蜂场的新出现的工蜂（Apis mellifera carnica）用3%草酸（OA）单独处理23。OA经常用于养蜂业，用于 瓦罗阿析构函数 控制。治疗后，将工蜂（每组三只）固定在冰上。解剖中肠并将其固定在10%福尔马林中。然后将组织在一系列酒精溶液中脱水，最后包埋在石蜡中。用切片机切成7μm薄片后，制?...

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

免疫组织化学方法可用于蜜蜂研究，以检测和评估成年蜜蜂中肠和下咽腺的细胞凋亡和坏死水平。

蜜蜂组织中的组织学基础和细胞死亡检测

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

该方案在用杀虫剂处理的蜜蜂的许多生物学和毒理学研究中很有用。它有助于检测杀螨剂蜜蜂组织的细胞反应，这些杀螨剂用于蜂群中用于治疗瓦螨。它是一种强大的工具，用于检测与蜜蜂或其他有益昆虫的行为相结合的对组织的亚小影响。首先，小心地用镊子取一只工蜂，将其放在零下 20 摄氏度的冰上或冰箱中两分钟以使其固定。将蜜蜂斜着固定在培养皿上，从左到右和从右到左两次穿过胸部的最上背部。可怜的昆虫盐水覆盖身体。将培养皿放在体视显微镜下，聚焦并调整。要解剖中肠，请从腹部开始，将剪刀的一个点插入蜜蜂身体右侧中心的 tergite A5 下方。切到特吉特 A2。保持剪刀内刃与身体侧面平行，以免损坏内脏。将剪刀向左转并剪。轻轻打开腹部的左侧并固定。用一只手用镊子轻轻向上拉蜜蜂胃，另一只手用剪刀在食道的最末端剪开。将胃和中肠从腹部拉开，并在直肠处切开。使用带有昆虫盐水溶液的移液管，并去除任何粪便或组织部分。为了解剖下咽腺，如前所述将工蜂固定在冰上。切下头部并将其放在较小的板上，天线朝上。用两个针固定头部，一个穿过左复眼，第二个穿过右复眼。在针脚内侧的第一个复眼上切开，继续切到盂唇，然后在另一侧穿过第二个复眼进行另一切口。切断触角。取下面膜，切下仍附着的地方。拿起镊子，小心地去除腺体以及大脑和部分复眼。对于苏木精和伊红染色，将脱蜡、再水化的切片放入苏木精中五分钟。然后，小心地将它们放在流动的自来水中两分钟。然后，将它们放入蒸馏水中一分钟，将伊红放入四分钟。将载玻片96%乙醇放置一分钟，然后放入2-丙醇两分钟，最后放入清除剂中两分钟。加入安装介质和盖玻片，让它们干燥。在光学显微镜下观察。准备科普林罐。准备蛋白酶-K。对切片进行脱蜡和再水化后，将载玻片放入PBS中五分钟。将载玻片平放在容器中并加入蛋白酶-K。用蒸馏水清洗载玻片。在室温下在内源性过氧化物酶中淬灭。用PBS或水冲洗载玻片。将载玻片平放在容器中，并在室温下应用平衡缓冲液10秒钟。擦拭组织周围后，向每个切片加入末端脱氧核苷酸转移酶，并在37摄氏度的加湿室中孵育一小时。孵育前，将湿纸巾放在载玻片周围的托盘内，并用保鲜膜覆盖。孵育后，将标本放在架子上，并将其留在停止洗涤缓冲液中。在PBS中清洗载玻片并在组织周围擦拭后，向切片中加入两滴温热的抗地高辛过氧化物酶偶联物，并在加湿的容器中孵育30分钟。在PBS中洗涤后，准备工作强度-过氧化物酶底物，用过氧化物酶底物覆盖切片，染色5分钟。将载玻片放在显微镜下并确定最佳染色时间。在染色架和蒸馏水中洗涤载玻片后，将载玻片在蒸馏水中孵育五分钟。使用苏木精复染两分钟。将载玻片放在流动的自来水中三分钟，然后在蒸馏水中清洗载玻片。将载玻片安装在玻璃盖玻片和安装介质下，平放晾干。在光学显微镜下观察。使用光学显微镜计算受影响细胞的百分比。结果表明，草酸处理显著影响中肠细胞。免疫染色显示下咽腺凋亡核中呈阳性红色或棕色反应产物。在大多数腺细胞中测定吡虫啉或香豆磷处理后的阳性反应产物。抬起蜂头面罩时，请注意不要损坏或拉下咽腺的部分。该技术可检测蜜蜂的亚临床变化，也适用于检测对蜜蜂和其他一些生物体的其他环境影响。

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.

肌动蛋白共沉淀测定;​​ F -肌动蛋白结合蛋白分析

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

科研

生物学

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.

脱钙骨组织学，形态计量学和荧光染料分析制备

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.

一个β-葡萄糖醛酸酶（GUS）的基细胞死亡含量

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.

使用LysoTracker检测及鉴别胚胎干细胞在胚胎细胞程序性死亡

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在原位</em&gt;改编为海洋软骨鱼类杂交整装胚胎发育和细胞组织学

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.

在出生后的小鼠听泡解剖：中耳骨骼和组织学分析的分离

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.

蛋白酶降解藻酸盐水凝胶和疏水性微生物反应器，用于猪卵母细胞封装

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.

使用基于多反应监测的人类脑组织蛋白质的多反应监测工作流程定量蛋白质组学工作流程

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

脂肪组织来源的干细胞分离和表征

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.

作者聚焦：分离和鉴定源自 Sprague Dawley 大鼠脂肪组织的间充质干细胞  

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

斯普拉格道利大鼠脂肪组织间充质干细胞的分离和鉴定

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