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

Jeg anerkender taknemmeligt støtten fra det slovenske forskningsagentur, bevilling nr. P4-133.

1. Grundlæggende histologi til honningbiforskning
<ol><li>Dissektion af honningbivæv BEMÆRK: Til dissektion af arbejderbier skal du bruge et dissekeringsmikroskop med en LED-lyskilde. Den mest nyttige forstørrelse er ~ 20x.<ol><li>Manipulation og dissektion<ol><li>Tag forsigtigt en arbejderbi med tang og læg den på is (eller i fryseren ved -20 °C) i 2 minutter for at immobilisere den22. Fastgør bien på petriskålen diagonalt gennem den øverste bageste del af brystka...

<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>

I levende organismer defineres celledød som apoptose eller nekrose25 og kan ledsages af autofagi26. Forskellen mellem apoptotiske og nekrotiske celler er, at apoptose er en form for programmeret celledød og forekommer i normale celler, mens nekrose opstår på grund af dødelige tilstande (f.eks. ulykke, sygdom)27,28. Apoptose kan detekteres ved hjælp af assaysæt baseret på TUNEL-teknikken (påvisning afDNA-fra...

Honningbier (Apis mellifera L.) er blandt andre vilde bestøvere de vigtigste bestøvere af landbrugsplanter. I løbet af tusinder af år har det skiftende miljø påvirket bier til at tilpasse deres morfologi, fysiologi, adfærd og tolerance til flere patogener og parasitter. Derfor har honningbier udviklet en meget forskelligartet vifte af arter og underarter rundt om i verden1. Disse resultater er i overensstemmelse med tidligere fund, at der er genetisk variation i honningbiens fordøjelseskanalstruktur, men tyder også på, at ændringer af midgut skyldes miljøfaktorer 2,3.
Honningbiens fordøjelseskanal har tre hoveddele: foregut, midgut (ventriculus) og hindgut4. Ventriculus er et vigtigt organ til fordøjelsen af pollen og nektar / honning; I bagkroppen foregår osmotisk kontrol gennem absorption af vand og ioner2. Hypopharyngeal kirtler (HPG'er) af honningbiarbejdere er placeret i hovedet og syntetiserer og udskiller kongelige gelékomponenter til at fodre bøden, dronningen og medlemmer af kolonien. Deres størrelse ændres med alder og opgaver og afhænger af korrekt ernæring (kvalitetspollen). Sygeplejersker i alderen 6 til 18 dage udfører yngelopdræt, og størrelsen på HPG'er stiger 5,6. I foragerbier degenererer HPG'erne og udskiller kun enzymer, der er vigtige for at omdanne de komplekse sukkerarter til enkle (α-glucosidaser, leucin arylamidase, invertase) i honning7.
Honningbier udsættes for flere biotiske og abiotiske stressfaktorer8, og fordøjelseskanalen kan påvirkes af flere negative stimulanser. Den første barriere, der beskytter organismen mod patogener, er den peritrofiske membran i midgut, som består af tarmslimhinden for at beskytte mod patogener4. Udviklingen og funktionen af HPG'er afhænger af kost, alder og kolonitilstand9 og påvirkes af insekticider, acaricider 10 og patogener11,12,13. Acaricide rester i bikuben på grund af varroa kontrol behandling og pesticider fra miljøet påvirker forager bier og sygeplejerske bier14,15. Den største trussel mod honningbikolonier er miden Varroa destructor, både som vektor af vira, der bidrager til kolonitab16 og som forbruger af værtens fedtlegeme (et vigtigt vitalt organ i honningbier), som følgelig påvirker individets krops- og kolonifunktioner17.
Imidlertid kan intensive landbrugsjordhabitater give en kortsigtet fødevareforsyning til honningbier. Derfor bør miljøvenlige landbrugsordninger øge tilgængeligheden af honningblomster i landbrugslandskaber18. For at vurdere morfologien af forskellige underarter 6,19,20,21 eller subletale virkninger af disse faktorer på celle- eller vævsniveau, især midgut og HPG'er, er histologiske og immunohistokemiske metoder praktiske og tilstrækkeligt nøjagtige til at blive anvendt i histologisk forskning i honningbier.

<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

Honningbier (Apis mellifera L.) inde i bikuben (sygeplejerskearbejdere og andre bikuber) og uden for bikuben (foragere) udsættes for klima- og vejrændringer, forskellige pesticider, patogener og underernæring, der hovedsageligt kommer ind gennem munden og primært påvirker fordøjelseskanalen hos voksne bier. For at forstå og forhindre virkningerne af sådanne eksterne og interne stressfaktorer på honningbier er en nyttig forskningsmetode den immunohistokemiske metode. En grundlæggende protokol er beskrevet for at forberede midgut (ventriculus) og hypopharyngeal kirtler (HPG'er) hos voksne bier til histologisk analyse. En detaljeret metode er beskrevet for at vurdere niveauet af celleskader og skelne nekrose fra programmeret celledød (apoptose) som en naturlig proces med vævsregenerering. Resultaterne af behandling af voksne honningbier med oxalsyre og pesticider (insekticid og acaricid) og bestemmelse af celledød i ventriculus og HPG'er præsenteres. Fordele og ulemper ved metoden diskuteres også.

Påvisning af celledød i midgut Nyopståede arbejderbier (Apis mellifera carnica) fra den eksperimentelle bigård ved Sloveniens landbrugsinstitut i Ljubljana blev individuelt behandlet med 3 % oxalsyre (OA)23. OA bruges ofte i biavl til Varroa destructor kontrol. Efter behandlingen blev arbejderbierne (tre fra hver gruppe) immobiliseret på is. Midgut blev dissekeret og fikseret det i 10% formalin. Vævet blev derefter dehydreret i en række alkoholopløsnin...

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

Immunohistokemiske metoder er nyttige i honningbiforskning til at detektere og vurdere niveauet af apoptose og nekrose i midgut og hypopharyngeal kirtler hos voksne bier.

Grundlæggende om histologi og påvisning af celledød i honningbivæv

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

Denne protokol er nyttig i mange biologiske og toksikologiske undersøgelser af honningbier behandlet med pesticider. Det hjælper med at detektere de cellulære reaktioner af honningbivæv af acaricider, der anvendes i bi-kolonier til behandling mod Varroa-mider. Det er et kraftfuldt værktøj til påvisning af sub-små effekter på væv i kombination med adfærd hos bier eller andre gavnlige insekter.Til at begynde med skal du forsigtigt tage en arbejderbi med tang og lægge den på is eller fryser ved minus 20 grader Celsius i to minutter for at immobilisere den. Fastgør bien på petriskålen diagonalt gennem den øverste bageste del af brystkassen to gange fra venstre mod højre og fra højre mod venstre. Dårlig insekt saltvand til at dække kroppen.Placer petriskålen under stereomikroskopet, fokuser og juster. For at dissekere midgut skal du starte med maven ved at indsætte et punkt på saksen under tergite A5 i midten af højre side af bi-kroppen. Klip til tergite A2. Hold saksens indre blad parallelt med siden af kroppen for at undgå at beskadige de indre organer.Drej saksen til venstre og klip. Åbn forsigtigt den venstre del af maven og fastgør den. Brug tang med den ene hånd til at trække honningbimaven forsigtigt opad og med en saks i den anden hånd skære i slutningen af spiserøret.Træk maven og midgut væk fra maven og skær ved endetarmen. Brug en pipette med insektsaltopløsning og fjern afføring eller dele af vævet. Til dissektion af hypopharyngeal kirtler, immobilisere en arbejdstagerbi på is som beskrevet før.Skær hovedet af og læg det på en mindre plade med antennen opad. Fastgør hovedet med to stifter, en gennem det venstre sammensatte øje og den anden gennem det højre sammensatte øje. Lav et snit over det første sammensatte øje på indersiden af stifterne, fortsæt til labrum, og lav derefter endnu et snit på den anden side over det andet sammensatte øje.Skær antennerne af. Løft masken af og klip, hvor den stadig er fastgjort. Tag tangene og fjern forsigtigt kirtlerne sammen med hjernen og en del af de sammensatte øjne.Til hæmatoxylin og eosin farvning sættes de afvoksede, rehydrerede sektioner i hæmatoxylin i fem minutter. Placer dem derefter forsigtigt under rindende ledningsvand i to minutter. Sæt dem derefter i destilleret vand i et minut og eosin i fire minutter.Anbring diasene 96% ethanol i et minut, derefter 2-propanol i to minutter og til sidst i clearingmidlet i to minutter. Tilsæt monteringsmedium og et dækglas og lad dem tørre. Overhold under et lysmikroskop.Forbered Coplin krukker. Forbered proteinase-K. Efter afvoksning og rehydrering af sektionerne skal du placere diasene i PBS i fem minutter.Placer diasene fladt i beholderen og tilsæt proteinase-K. Vask diasene i destilleret vand. Sluk i endogen peroxidase ved stuetemperatur.Skyl rutsjebanerne med PBS eller vand. Placer diasene fladt i beholderen, og påfør ækvilibreringsbuffer i 10 sekunder ved stuetemperatur. Efter aftørring omkring vævet tilsættes det terminale deoxynukleotidyltransferaseenzym til hver sektion og inkuberes i et befugtet kammer i en time ved 37 grader Celsius.Før inkubation skal du lægge fugtede papirhåndklæder inde i bakken omkring diasene og dække dem med plastfolie. Efter inkubation sættes prøverne på stativet og efterlades i stop-wash buffer. Efter vask af diasene i PBS og aftørring omkring vævene tilsættes to dråber varm anti-digoxigeninperoxidasekonjugat til sektionerne og inkuberes i 30 minutter i en befugtet beholder.Efter vask i PBS skal du forberede arbejdsstyrkeperoxidasesubstrat, dække sektionerne med peroxidasesubstrat og plette i fem minutter. Placer diaset under mikroskopet og bestem den optimale farvningstid. Efter vask af diasene i et farvningsstativ og destilleret vand, inkuberes diasene i destilleret vand i fem minutter.Modplet ved hjælp af hæmatoxylin i to minutter. Vask rutsjebanerne ved at placere dem under rindende ledningsvand i tre minutter og derefter i destilleret vand. Monter diasene under glasdæksler og monteringsmedium, og lad det tørre det tørre.Overhold under et lysmikroskop. Procentdelen af berørte celler blev beregnet ved hjælp af et lysmikroskop. Resultaterne viste, at behandlingen med oxalsyre signifikant påvirkede cellerne i midgut.Immunostaining viste positive røde eller brune reaktionsprodukter i de apoptotiske kerner af hypopharyngeale kirtler. Positivt reaktionsprodukt efter behandling med imidacloprid eller coumaphos blev bestemt i de fleste kirtelceller. Når du løfter masken på bihovedet, skal du passe på ikke at beskadige eller trække dele af hypopharyngealkirtlerne af.Denne teknik registrerer subkliniske ændringer i bier og er også velegnet til påvisning af andre miljøpåvirkninger på bier og nogle andre levende organismer.

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.

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

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.

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.

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.

Brug af LysoTracker at opdage programmeret celledød i Embryoner og Differentiering embryonale stamceller

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; In situ</em&gt; Hybridisering i Hele Mount Embryoner og Cell Histologi Tilpasset til Marine bruskfisk

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.

Dissektion af Auditory Bulla i postnatale mus: Isolering af mellemøret Knogler og Histologisk analyse

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.

Proteolytisk nedbrudt alginathydrgels og hydrofobe mikrobioreaktorer til Porcine Oocyt Indkapsling

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.

Kvantitativ Proteomics Workflow ved hjælp af flere reaktionsovervågningsbaserede detektioner af proteiner fra humant hjernevæv

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

Fedtvævsafledt stamcelleisolering og karakterisering

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.

Forfatter Spotlight: Isolering og identifikation af mesenkymale stamceller afledt af fedtvæv fra Sprague Dawley-rotter  

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

Isolering og identifikation af mesenkymale stamceller afledt af fedtvæv fra Sprague Dawley rotter

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