We would like to acknowledge our colleague Dr. Jalaiah Varikooty who provided insight and expertise on the topic of the lid wiper, that greatly assisted this research.

Etik deyimi: Ön insan denekler hücreleri toplamak için, aydınlatılmış onam ve etik onayı alınmalıdır. 1. Leke hazırlayın NOT: Deney günü boya hazırlayın. <ol><li> 15 ml santrifüj tüpü içinde 2.5 ml fosfat tamponlu tuz (PBS) ile 5 ul Etidyum (veya 4 uM) ve 5 ul Calcein AM (veya 4 uM) ilave et; karıştırın. kullanılana kadar oda sıcaklığında hafif ve deposundan korumak için alüminyum folyo ile sarılır. </li></ol> 2. Toplama Hücreler <ol><li> kendi paketinden hücre kültürü ekler çıkarın ve her göz kapağı numune alınacak için etiket. kalıcı bir kalem kullanarak zar plastik tutucu tarafında hücre toplama Mark yönelimi. Davranış IC geçmesi bölgenin uygun sağlığını onaylamak için orta büyütme (yaklaşık 20X) Lambanın denetim yarık. </li><li> ea alt konjunktival kesesine topikal anestezi (% 0.5 proparacaine hidroklorür) bir damla dağıtınch göz ve gözleri korumak için katılımcıyı talimat bir dakika süreyle kapattı. </li><li> Evert üst göz kapağı ve kirpik tarafından yerinde tutun; kapak silecek alanı ile herhangi bir temastan kaçının. İsteğe bağlı: İkincil araştırmacı yardım Bu adımı gerçekleştirmek gerekebilir. </li><li> minimal basınç ile kapak silecek bölgenin orta kısmında dik olarak membran uygulayın. </li><li> 5 saniye - 3 yerine membran tutun. iletişim alanında saydam hale membran gözlemleyin. Bu noktada, hafifçe membran kaldırmak ve yerine geri &quot;çevirmek&quot; için katılımcının kapağını izin verir. </li><li> Derhal kurumasını önlemek için, örnek PBS bir damla uygulanır. Membran saydam dönecek ve bu kurumayı belirttiği gibi, herhangi bir zamanda opak dönmemelidir. İkincil araştırmacı örneklerinin işlenmesi (bölüm 3) ile hemen devam var. </li><li> konjonktiva bütünlüğünü sağlamak için nihai göz kontrolü yapıyoruz. oküler yağlayıcılar koyun ve katılımcıyı talimatanestetik etki kapalı giyer kadar gözlerini ovuşturarak önlemek için (yakl. 15 dk). </li></ol> 3. Örnek İşleme <ol><li> mikro-makas kullanılarak, dikey hücresi ile toplama ortasından ikiye (alt analizler her biri için bir adet yarım) membran kesti. plastik tutucu ayırmak için bir membran yarısının dış kenarı boyunca kesme; membran kendi üzerine kat etmemesini sağlamak da dahil olmak üzere membran üzerinde toplanan hücreleri ile etkileşimi önlemek. </li><li> tamamen sahibinden ayrılmakta önce cımbız kullanarak membran sabitleyin. % 95 (h / h) ihtiva eden bir 500 ul ihtiva eden ve (Bölüm 4.2) işleme önce 2 saat, en fazla 20 dakika içinde en az düzeltmek için terk etiketli 2 ml santrifüj tüpüne yer membran. </li><li> (Bölüm 3.2&#39;de anlatıldığı gibi) ve derhal bölüm 4.1 ile devam membran hemen ayrı ikinci yarısı. </li></ol> 4. Numune Boyama <ol><li> Immunocytochemical Boyama <ol><li> Dikkatle toplama yüzü aşağı bakacak şekilde 35 mm cam alt kültür çanak membran yerleştirin; Membran düz olduğundan emin olun. </li><li> Pipet zara bölüm 1 monte İmmünositokimyasal leke kompozisyon 20 ul. Örnek bukleler kadar, dikkatle kenarlarını bastırarak membran düzleştirmek için tek kullanımlık pipet uçları kullanın. Hücre toplama alanları ile temastan kaçının. </li><li> Yavaşça cam kapak slip ile membran kapak. Numunenin gereksiz hareketini önlemek. Kapaklı çanak Kapak ve kenarlarda laboratuvar film ile kapatın. çanak (üstte ve altta) ve numunenin görünürlük şeffaflık engellemeyin. laboratuar işaretleyici ile etiketleyin. </li><li> Hemen görüntüleme (bölüm 5.1) ile devam, sonra membran diğer yarısı sitolojik boyama ile devam etmek için bölüm 4.2 dönün. </li></ol></li><li> sitolojik Boyama <ol><li> Yavaş yavaş yavaş tüp u damıtılmış su, 500 ul ekleyerek membran hidratbölüm 3.4 tespiti için sed. pipet içine aspire içeriği defalarca sonra içeriğini kaldırmak, karıştırın. </li><li> damıtılmış su, 500 ul ekle. Birkaç saniye için ayakta Izin, sonra çıkarın. Alcian blue 500 ul ilave edin ve 3 dakika boyunca bırakın. leke çıkarın ve 3 ardışık 500 ul damıtılmış su durular veya sıvı berrak durular kadar gerçekleştirin. </li><li> Hematoksilen 1. leke 500 ul ekleyin ve 3 dakika bekletin. leke çıkarın ve üç ardışık 500 ul distile su durulama gerçekleştirmek veya sıvı berrak durular kadar. berrak bir sıvı aspire (bölüm 4.2.1 tersini) kurutmak. </li><li> Papanicolaou RG-6 leke 500 ul ekleyin ve 3 dakika bekletin. leke çıkarın ve bir 500 ul% 95 (v / v) etanol durulama gerçekleştirin. </li><li> Papanicolaou EA-65 boyasının 500 ul ekleyin ve 3 dakika bekletin. leke çıkarın ve 3 ardışık 500 ul% 95 yerine (v / v) etanol durular. </li><li> Tam üç ardışık 500 ul% 100 etanol durulama kullanarak kurutmak. TWE kullanarakezers, çözeltiden örneği kaldırmak; dokunaklı hücre toplama alanları kaçının. </li><li> cam slayt membran yerleştirin; Bu hücre toplama hattı sağlamak paralel veya görüntüleme sırasında daha kolay uyum için marjını slayt dik olarak ayrılmaktadır. </li><li> % 100 etanol bir damla uygulayın ve cam slip ile kaplayın. Hemen görüntüleme (bölüm 5.2) ile devam edin. </li></ol></li></ol> 5. Numune Görüntüleme <ol><li> Görüntüleme Floresan İmmünositokimyasal Renklendiriciler <ol><li> Bir görüntü elde etme yazılımı çalıştıran bir bilgisayara bağlı bir dijital renkli kamera ile donatılmış bir konfokal lazer tarama mikroskobu (CLSM) 11 veya floresan mikroskop 12, kullanın. örnekler kültür tabaklarına içinde bulunan kısmi olarak, bir inverted mikroskop gerekli olabilir. </li><li> Etidyum homodimer-1 (DNA&#39;nın mevcudiyetinde 528/617 nm örn / Em maxima) ve kalsein AM absorpsiyon ve emisyon spektrumu (494/517 nm e göre olan mikroskop 11,12 ayarlamax / Em maxima). </li><li> (Örneğin, 2.5X objektif) mikroskop düşük büyütme seçin ve dijital görüntüleme sistemini aktive; bilgisayar ekranında görüntülenen örnek gözlemlemek. </li><li> ilgi hücresel özellikleri için örnek olup olmadığını kontrol edin. </li><li> İstenilen mikroskop büyütme seçin ve görüntüleme yazılımı kullanarak görüntüleri kazanır. </li><li> Yerli mikroskop dosya biçiminde veya sıkıştırılmamış bir dosya biçiminde (örneğin, * .raw, * .tiff) ya dosyaları kaydedin. </li></ol></li><li> Görüntüleme Sitolojik Lekeleri <ol><li> Bir görüntü elde etme yazılımı çalıştıran bir bilgisayara bağlı bir dijital renkli kamera ile donatılmış herhangi bir filtre standart bir laboratuvar parlak alan mikroskobu, kullanın. </li><li> Yeri ve mikroskop sahnede (adım 4.2.8 itibaren) kilit cam slayt. gerektiği gibi tüm görüntüleme işlem boyunca,% 100 etanol ile örnek rehidrate. </li><li> (Örneğin, 2.5X objektif) mikroskop düşük büyütme seçin ve inci etkinleştirmekE dijital görüntüleme sistemi; bilgisayar ekranında görüntülenen örnek gözlemlemek. </li><li> Emin olun hücre toplama mikroskop sahne kontrolü X veya Y ekseni ya uyumludur. kapak kayma kaldırarak ve bir pipet veya cımbız kullanarak örnek çevirerek, gerekirse ayarlayın. </li><li> görüntüleme yazılımı, en düşük mikroskop büyütme ile tüm numunenin yakalama resim (ler) kullanma. </li><li> Orta mikroskop büyütme geçin (örneğin, 10 - 20X objektif) ve mikroskop ışık kaynağı ve yazılım görüntüleme parametrelerinin (pozlama, kontrast, beyaz dengesi, vs.) ayarlayabilir ve onların otomatik yazılım ölçüm devre dışı bırakın. ileride kullanmak için bu ayarları kaydedin. </li><li> Tarama (örneğin, sol üst ve hücre koleksiyonu sağ alt köşelerinde) başlangıç ​​ve bitiş noktalarını belirleyin. </li><li> görüntüleme yazılımı, başlangıç ​​ve bitiş noktaları içinde tüm hücre toplama alanının yakalama resim (ler) kullanma. % 20 yan-yana ve üst-to-alt üst üste betw sağlamaktüm bitişik görüntüleri een. </li><li> Yerli mikroskop dosya biçimini veya sıkıştırılmamış dosya biçimi (örneğin, * .raw, * .tiff) kullanarak dosyaları kaydedin. </li><li> (Örneğin, Adobe Photoshop Elements) seçim otomatik dikiş yazılımını kullanarak nihai panoramik görüntü oluşturun. Son görüntüde otomatik dikiş doğruluğunu onaylamak için adım 5.2.5 alınan referans görüntüleri) kullanın. </li></ol></li></ol>

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The authors have no relevant funding or conflicts to disclose.

Gösterim sitoloji genellikle karışık selüloz ester membranlar kullanılarak, konjonktivada üzerinde gerçekleştirilir. Numuneler, dökme malzeme levhalar boyutta kesilir sterilize forseps kullanılarak konjunktival yüzeye uygulanan ve kaldırılır. Aynı membran malzeme kullanarak, bir piston kontrollü IC cihaz son zamanlarda uygulamalar 13 arasında tutarlı bir baskı bulber konjonktiva yüzey geometrisini maç ve korumak için tasarlanmıştır. Bu yaklaşımlar bölge silecek dar, belirgin kavis...

2 mm dar konjonktiva bölge göz küre karşı - insan üst göz kapağı sadece 1 ile 10,000 yanıp her gün yaklaşık 1 yürütür. Yanıp sırasında silme hareketi nedeniyle, göz kapağı kenarında bu bölümü &quot;kapak silecek&quot; bölgesini 2 olarak adlandırılmıştır. Sürtünme yüzünden dünya üzerinde silme kapak, alışılmış kırpma sırasında bu bölgede yüksek olduğu varsayılmaktadır. Bu göz konfor önemli bir rol oynayabilir. Kontakt lens özellikle deneyim göz rahatsızlık kullananlar ve bu mercek 3 giymek durdurması için birincil nedenlerinden biridir. Bir kontakt lens göze yerleştirildiğinde, lens materyali ve göz yüzeyi arasındaki sürtünme katsayısı 4 değiştirme gösterilmiştir. Kuruluk belirtileri bu değiştirilmiş sürtünme 2,5,6 ilişkili olabileceğini gösteren kanıtlar vardır. Bu ilişki aynı zamanda klinik olarak gözlemlenebilir olayların yansıması olabilir, özellikle kapak wiepitelyopati (LWE) da denilen üst kapak marjı boyama (ULMS) 6 başına. LWE oküler irritasyon erken bir belirtisi ve kuru göz hastalığı için potansiyel bir belirleyicisidir. Bu görülen üst ve alt kapak kenarında bölgelerinin önemli bir boyama ile ölçülür. Bu derecelendirme sistemi 2 ve klinik geçerliliği (yani öznel belirtiler ile korelasyon) tartışma halen iken, göz rahatsızlığı, hem doktorlar ve bilim adamları için bir bilmece kalır ve en önemlisi, hastalar için bir rahatsızlık. To-date, küçük kapak silecek alanının 7 (alt), hücresel anatomi ve fizyoloji klinik-alakalı varyasyonları hakkında bilinmektedir ve sadece tek bir rapor sitolojik yöntemler 8 kullanmaktadır. Gösterim sitoloji (IC) bir zar 9,10 uygulanması ile bulber veya tarsal konjonktiva epitel yüzeyinden hücreleri toplamak için 40 yıldır istihdam edilmiştir. çıkarılması üzerine, yapışan hücreler c geçmesiytological boyama ve mikroskopik görüntü. Nedeniyle kapak silecek bölgedeki anatomik ve hücresel farklılıkları, bu teknik optimizasyon gerektirir.

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			<td height="20" style="height:20px;width:349px;">Alcian Blue, 1% in 3% Acetic Acid</td>
			<td style="width:119px;">Sigma</td>
			<td style="width:119px;">B8438-250ML</td>
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			<td height="20" style="height:20px;">Hematoxylin, Gill 1</td>
			<td style="width:119px;">Sigma</td>
			<td>GHS116-500ML</td>
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			<td height="20" style="height:20px;">Papanicolaou stain, OG-6</td>
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			<td>HT40116-500 ml</td>
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			<td height="20" style="height:20px;">Papanicolaou stain, Modified EA</td>
			<td style="width:119px;">Sigma</td>
			<td>HT40232-1L</td>
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			<td height="20" style="height:20px;">Live/Dead Viability/Cytotoxicity Kit</td>
			<td>Life Technologies</td>
			<td>L-3224</td>
			<td>contains ethidium homodimer-1 and Calcein AM dyes</td>
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			<td height="20" style="height:20px;">Alcaine (0.5% proparacaine hydrochloride)</td>
			<td>Alcon</td>
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			<td height="20" style="height:20px;">Millicell Cell Culture Insert, 12&#160;mm, hydrophilic PTFE, 0.4&#160;&#181;m</td>
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			<td>PICM01250&#160;</td>
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			<td height="20" style="height:20px;">35 mm Glass Bottom Dishes</td>
			<td>MatTek Corporation</td>
			<td>P35G-0-20-C</td>
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impression cytology of the lid wiper area

Few reports on the cellular anatomy of the lid wiper (LW) area of the inner eyelid exist and only one report makes use of cytological methods. The optimization of a method of collecting, staining and imaging cells from the LW region using impression cytology (IC) is described in this study. Cells are collected from the inner surface of the upper eyelid of human subjects using hydrophilic polytetrafluoroethylene (PTFE) membranes, and stained with cytological dyes to reveal the presence of goblet cells, mucins, cell nuclei and various degrees of pre- and para-keratinization. Immunocytochemical dyes show cell esterase activity and compromised cell membranes by the use of a confocal scanning laser microscope. Up to 100 microscopic digital images are captured for each sample and stitched into a high-resolution, large scale image of the entire IC span. We demonstrate a higher sensitivity of IC than reported before, appropriate for identifying cellular morphologies and metabolic activity in the LW area. To our knowledge, this is the first time this selection of fluorescent dyes was used to image LW IC membranes. This protocol will be effective in future studies to reveal undocumented details of the LW area, such as assessing cellular particularities of contact lens wearers or patients with dry eye or lid wiper epitheliopathy.

plastik tutucu üzerine yayılmış hücre kültürü membran kapak silecek konjonktiva epitel hücrelerinin el toplanması için uygun bir araçtır. Bu yöntem genellikle hazırlamak ve IC membranlar işlemek için kullanılan ek sterilize aletler için gereğini ortadan kaldırır. 1 hücre kültürü eki kullanarak kapak silecek alanının IC tasvir Şekil. Biz yeni bir moda kapak silecek alanından epitel hücreleri karakterize etmek için birbirini...

Watch this Scientific Journal Video about Impression Cytology of the Lid Wiper Area at JoVE.com

Impression Cytology of the Lid Wiper Area

We collected, stained and imaged cells from the conjunctiva of the inner eyelid margin of human subjects. By characterizing cell morphology and metabolic activity, this method may further our understanding of dry eye and the role that friction between the ocular surfaces may play in perceiving ocular discomfort.

Kapak Silecek Area izlenim Sitoloji

Few reports on the cellular anatomy of the lid wiper (LW) area of the inner eyelid exist and only one report makes use of cytological methods. The ...

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JoVE Journal

Medicine

10.3K Görüntüleme.  University of Waterloo. We collected, stained and imaged cells from the conjunctiva of the inner eyelid margin of human subjects. By characterizing cell morphology and metabolic activity, this method may further our understanding of dry eye and the role that friction between the ocular surfaces may play in perceiving ocular discomfort. 

Video: Impression Cytology of the Lid Wiper Area

Detection and Genogrouping of Noroviruses from Children's Stools By Taqman One-step RT-PCR

Noroviruses (NoVs) are the leading cause of outbreaks of sporadic acute gastroenteritis worldwide in humans of all ages. They are important cause of hospitalizations in children with a public health impact similar to that of Rotavirus. NoVs are RNA viruses of great genetic diversity and there is a continuous appearance of new strains. Five genogroups are recognized; GI and GII with their many genotypes and subtypes being the most important for human infection. However, the diagnosis of these two genotypes remains problematic, delaying diagnosis and treatment. 1, 2, 3
For RNA extraction from stool specimens the most commonly used method is the QIAmp Viral RNA commercial kit from Qiagen. This method combines the binding properties of a silica gel membrane, buffers that control RNases and provide optimum binding of the RNA to the column together with the speed of microspin. This method is simple, fast and reliable and is carried out in a few steps that are detailed in the description provided by the manufacturer. 
 Norovirus is second only to rotavirus as the most common cause of diarrhea. Norovirus diagnosis should be available in all studies on pathogenesis of diarrhea as well as in outbreaks or individual diarrhea cases. At present however norovirus diagnosis is restricted to only a few centers due to the lack of simple methods of diagnosis. This delays diagnosis and treatment 1, 2, 3. In addition, due to costs and regulated transportation of corrosive buffers within and between countries use of these manufactured kits poses logistical problems. As a result, in this protocol we describe an alternative, economic, in-house method which is based on the original Boom et al. method4 which uses the nucleic acid binding properties of silica particles together with the anti-nuclease properties of guanidinium thiocyanate.
For the detection and genogrouping (GI and GII) of NoVs isolates from stool specimens, several RT-PCR protocols utilizing different targets have been developed. The consensus is that an RT-PCR using TaqMan chemistry would be the best molecular technique for diagnosis, because it combines high sensitivity, specificity and reproducibility with high throughput and ease of use. Here we describe an assay targeting the open reading frame 1 (ORF1)-ORF2 junction region; the most conserved region of the NoV genome and hence most suitable for diagnosis. For further genetic analysis a conventional RT-PCR that targets the highly variable N-terminal-shell from the major protein of the capsid (Region C) using primers originally described by Kojima et al. 5 is detailed. Sequencing of the PCR product from the conventional PCR enables the differentiation of genotypes belonging to the GI and GII genogroups.

A One-Step RT-PCR assay for detection and genogroup identification of Norovirus isolates from children&#x2019;s stools, that utilizes primers and TaqMan probes specific to the open reading frame 1 (ORF1)-ORF2 junction region, the most conserved region of the Norovirus genome is described. A non-commercial, cost-effective RNA extraction method is detailed.

Taqman By Çocuk Tabureler gelen Noroviruses tespiti ve Genogrouping Tek aşamalı RT-PCR

Noroviruses (NoVs) are the leading cause of outbreaks of sporadic acute gastroenteritis worldwide in humans of all ages. They are important cause of ...

Tıp

Immunohistochemical Staining of B7-H1 (PD-L1) on Paraffin-embedded Slides of Pancreatic Adenocarcinoma Tissue

B7-H1/PD-L1, a member of the B7 family of immune-regulatory cell-surface proteins, plays an important role in the negative regulation of cell-mediated immune responses through its interaction with its receptor, programmed death-1 (PD-1) 1,2. Overexpression of B7-H1 by tumor cells has been noted in a number of human cancers, including melanoma, glioblastoma, and carcinomas of the lung, breast, colon, ovary, and renal cells, and has been shown to impair anti-tumor T-cell immunity3-8.
Recently, B7-H1 expression by pancreatic adenocarcinoma tissues has been identified as a potential prognostic marker9,10. Additionally, blockade of B7-H1 in a mouse model of pancreatic cancer has been shown to produce an anti-tumor response11. These data suggest the importance of B7-H1 as a potential therapeutic target. Anti-B7-H1 blockade antibodies are therefore being tested in clinical trials for multiple human solid tumors including melanoma and cancers of lung, colon, kidney, stomach and pancreas12.
In order to eventually be able to identify the patients who will benefit from B7-H1 targeting therapies, it is critical to investigate the correlation between expression and localization of B7-H1 and patient response to treatment with B7-H1 blockade antibodies. Examining the expression of B7-H1 in human pancreatic adenocarcinoma tissues through immunohistochemistry will give a better understanding of how this co-inhibitory signaling molecule contributes to the suppression of antitumor immunity in the tumor's microenvironment. The anti-B7-H1 monoclonal antibody (clone 5H1) developed by Chen and coworkers has been shown to produce reliable staining results in cryosections of multiple types of human neoplastic tissues4,8, but staining on paraffin-embedded slides had been a challenge until recently13-18. We have developed the B7-H1 staining protocol for paraffin-embedded slides of pancreatic adenocarcinoma tissues. The B7-H1 staining protocol described here produces consistent membranous and cytoplasmic staining of B7-H1 with little background.

Immunohistochemical Staining of B7-H1 PD-L1 on Paraffin-embedded Slides of Pancreatic Adenocarcinoma Tissue

B7-H1 (PD-L1) and its binding to PD-1 provide a major tumor-induced immunosuppressive signal in the tumor&#x2019;s microenvironment. An immunohistochemical staining technique to characterize the expression and localization of B7-H1 in pancreatic adenocarcinoma is described here.

Pankreas Adenokarsinom Doku B7-H1 immünohistokimyasal boyanma (PD-L1) parafin Slaytlar

B7-H1/PD-L1, a member  of the B7 family of immune-regulatory cell-surface proteins, plays an important  role in the negative regulation of cell-mediated ...

NADH Fluorescence Imaging of Isolated Biventricular Working Rabbit Hearts

Since its inception by Langendorff1, the isolated perfused heart remains a prominent tool for studying cardiac physiology2. However, it is not well-suited for studies of cardiac metabolism, which require the heart to perform work within the context of physiologic preload and afterload pressures. Neely introduced modifications to the Langendorff technique to establish appropriate left ventricular (LV) preload and afterload pressures3. The model is known as the isolated LV working heart model and has been used extensively to study LV performance and metabolism4-6. This model, however, does not provide a properly loaded right ventricle (RV). Demmy et al. first reported a biventricular model as a modification of the LV working heart model7, 8. They found that stroke volume, cardiac output, and pressure development improved in hearts converted from working LV mode to biventricular working mode8. A properly loaded RV also diminishes abnormal pressure gradients across the septum to improve septal function. Biventricular working hearts have been shown to maintain aortic output, pulmonary flow, mean aortic pressure, heart rate, and myocardial ATP levels for up to 3 hours8.
When studying the metabolic effects of myocardial injury, such as ischemia, it is often necessary to identify the location of the affected tissue. This can be done by imaging the fluorescence of NADH (the reduced form of nicotinamide adenine dinucleotide)9-11, a coenzyme found in large quantities in the mitochondria. NADH fluorescence (fNADH) displays a near linearly inverse relationship with local oxygen concentration12 and provides a measure of mitochondrial redox state13. fNADH imaging during hypoxic and ischemic conditions has been used as a dye-free method to identify hypoxic regions14, 15 and to monitor the progression of hypoxic conditions over time10.
The objective of the method is to monitor the mitochondrial redox state of biventricular working hearts during protocols that alter the rate of myocyte metabolism or induce hypoxia or create a combination of the two. Hearts from New Zealand white rabbits were connected to a biventricular working heart system (Hugo Sachs Elektronik) and perfused with modified Krebs-Henseleit solution16 at 37 &deg;C. Aortic, LV, pulmonary artery, and left &amp; right atrial pressures were recorded. Electrical activity was measured using a monophasic action potential electrode. To image fNADH, light from a mercury lamp was filtered (350&plusmn;25 nm) and used to illuminate the epicardium. Emitted light was filtered (460&plusmn;20 nm) and imaged using a CCD camera. Changes in the epicardial fNADH of biventricular working hearts during different pacing rates are presented. The combination of the heart model and fNADH imaging provides a new and valuable experimental tool for studying acute cardiac pathologies within the context of realistic physiological conditions.

The objective is to monitor the mitochondrial redox state of isolated hearts within the context of physiologic preload and afterload pressures. A biventricular working rabbit heart model is presented. High spatiotemporal resolution fluorescence imaging of NADH is used to monitor the mitochondrial redox state of epicardial tissue.

İzole Biventriküler ve NADH Floresans Görüntüleme Tavşan Kalpler Çalışma

Since its inception by Langendorff1, the isolated perfused heart remains a prominent tool for studying cardiac physiology2. However, it is not well-suited ...

Microarray-based Identification of Individual HERV Loci Expression: Application to Biomarker Discovery in Prostate Cancer

The prostate-specific antigen (PSA) is the main diagnostic biomarker for prostate cancer in clinical use, but it lacks specificity and sensitivity, particularly in low dosage values1&#8203;&#8203;. &#8216;How to use PSA&#39; remains a current issue, either for diagnosis as a gray zone corresponding to a concentration in serum of 2.5-10 ng/ml which does not allow a clear differentiation to be made between cancer and noncancer2 or for patient follow-up as analysis of post-operative PSA kinetic parameters can pose considerable challenges for their practical application3,4. Alternatively, noncoding RNAs (ncRNAs) are emerging as key molecules in human cancer, with the potential to serve as novel markers of disease, e.g. PCA3 in prostate cancer5,6 and to reveal uncharacterized aspects of tumor biology. Moreover, data from the ENCODE project published in 2012 showed that different RNA types cover about 62% of the genome. It also appears that the amount of transcriptional regulatory motifs is at least 4.5x higher than the one corresponding to protein-coding exons. Thus, long terminal repeats (LTRs) of human endogenous retroviruses (HERVs) constitute a wide range of putative/candidate transcriptional regulatory sequences, as it is their primary function in infectious retroviruses. HERVs, which are spread throughout the human genome, originate from ancestral and independent infections within the germ line, followed by copy-paste propagation processes and leading to multicopy families occupying 8% of the human genome (note that exons span 2% of our genome). Some HERV loci still express proteins that have been associated with several pathologies including cancer7-10. We have designed a high-density microarray, in Affymetrix format, aiming to optimally characterize individual HERV loci expression, in order to better understand whether they can be active, if they drive ncRNA transcription or modulate coding gene expression. This tool has been applied in the prostate cancer field (Figure 1).

Human endogenous retroviruses (HERV), which occupy 8% of the human genome, retain scarce coding capacities but a hundred thousand long terminal repeats (LTRs). A custom Affymetrix microarray was designed to identify individual HERV locus expression and was used on prostate cancer tissues as a proof of concept for future clinical studies.

Prostat Kanserinde Biyomarker Discovery Uygulama: Bireysel herv Lokusları İfade Mikroarray tabanlı Kimlik

The prostate-specific antigen (PSA) is the main diagnostic biomarker for prostate cancer in clinical use, but it lacks specificity and sensitivity, ...

Collection and Extraction of Saliva DNA for Next Generation Sequencing

The preferred source of DNA in human genetics research is blood, or cell lines derived from blood, as these sources yield large quantities of high quality DNA. However, DNA extraction from saliva can yield high quality DNA with little to no degradation/fragmentation that is suitable for a variety of DNA assays without the expense of a phlebotomist and can even be acquired through the mail. However, at present, no saliva DNA collection/extraction protocols for next generation sequencing have been presented in the literature. This protocol optimizes parameters of saliva collection/storage and DNA extraction to be of sufficient quality and quantity for DNA assays with the highest standards, including microarray genotyping and next generation sequencing.

DNA extraction from saliva can provide a readily available source of high molecular weight DNA, with little to no degradation/fragmentation. This protocol provides optimized parameters for saliva collection/storage and DNA extraction to be of sufficient quality and quantity for downstream DNA assays with high quality requirements.

Yeni Nesil dizinleri için Toplama ve Tükrük DNA çıkarımı

The preferred source of DNA in human genetics research is blood, or cell lines derived from blood, as these sources yield large quantities of high quality ...

A "Patient-Like" Orthotopic Syngeneic Mouse Model of Hepatocellular Carcinoma Metastasis

The majority of cancer-related deaths are caused by the metastasis of the cancer rather than the primary tumor itself. Yet, the underlying mechanisms of cancer metastasis are still unclear. Animal models are essential for elucidating the mechanisms and for evaluating novel strategies for the treatment of metastatic cancers. Here, an in-depth description of a &#8220;patient-like&#8221; orthotopic syngeneic mouse model for exploring the mechanisms of metastasis of solid organ tumors is provided. The survival surgical implantation of BNL 1ME A.7R.1 mouse hepatocellular carcinoma cells directly into the liver (the organ of origin) of the inbred wild-type immune competent laboratory mouse strain, BALB/c is described. The success and reproducibility of this methodology recommends it for widespread use in elucidating the biological mechanisms of solid organ cancer metastasis.

The metastatic spread of cancer is the major cause of cancer-related deaths. We provide an in-depth description of our survival surgery methodology for establishing a &#8220;patient-like&#8221; orthotopic syngeneic mouse model system for studying the mechanisms of metastasis in solid organ tumors.

Bir &quot;Hasta gibi&quot; Ortotopik singenik Fare Modeli Hepatoselüler karsinomu metastazı

The majority of cancer-related deaths are caused by the metastasis of the cancer rather than the primary tumor itself. Yet, the underlying mechanisms of ...

Induction of Ischemic Stroke and Ischemia-reperfusion in Mice Using the Middle Artery Occlusion Technique and Visualization of Infarct Area

Cerebrovascular disease is highly prevalent in the global population and encompasses several types of conditions, including stroke. To study the impact of stroke on tissue injury and to evaluate the effectiveness of therapeutic interventions, several experimental models in a variety of species were developed. They include complete global cerebral ischemia, incomplete global ischemia, focal cerebral ischemia, and multifocal cerebral ischemia. The model described in this protocol is based on the middle cerebral artery occlusion (MCAO) and is related to the focal ischemia category. This technique produces consistent focal ischemia in a strictly defined region of the hemisphere and is less invasive than other methods. The procedure described is performed on mice, given the availability of several genetic variants and the high number of tests standardized for mice to aid in the behavioral and neurodeficit evaluation.

We describe a mouse model of stroke induced by the occlusion of the middle cerebral artery using a silicone coated suture. The protocol can be applied to induce permanent occlusion or a temporary ischemia, followed by reperfusion.

Enfarktüs Bölgesi Orta Arter Tıkanıklığı Tekniği ve Görselleştirme kullanma Fare İskemik İnme ve İskemi-reperfüzyon indüksiyon

Cerebrovascular disease is highly prevalent in the global population and encompasses several types of conditions, including stroke. To study the impact of ...

Automated Radiochemical Synthesis of [18F]3F4AP: A Novel PET Tracer for Imaging Demyelinating Diseases

3-[18F]fluoro-4-aminopyridine, [18F]3F4AP, is a radiofluorinated analog of the FDA-approved drug for multiple sclerosis 4-aminopyridine (4AP). This compound is currently under investigation as a PET tracer for demyelination. We recently described a novel chemical reaction to produce metafluorinated pyridines consisting of direct fluorination of a pyridine N-oxide and the utilization of this reaction for the radiochemical synthesis of [18F]3F4AP. In this article, we demonstrate how to produce this tracer using an automated synthesizer and an in-house made flow hydrogenation reactor. We also show the standard quality control procedures performed before releasing the radiotracer for preclinical animal imaging studies. This semi-automated procedure may serve as the basis for future production of [18F]3F4AP for clinical studies.

Automated Radiochemical Synthesis of [18F]3F4AP: A Novel PET Tracer for Imaging Demyelinating Diseases

We demonstrate the semi-automated radiochemical synthesis of [18F]3F4AP and quality control procedures.

Otomatik Radyokimyasal Sentez [<sup&gt; 18</sup&gt; F] 3F4AP: Demyelinizan Hastalıkların Görüntülenmesine Yönelik Yeni Bir PET İzleyici

3-[18F]fluoro-4-aminopyridine, [18F]3F4AP, is a radiofluorinated analog of the FDA-approved drug for multiple sclerosis 4-aminopyridine (4AP). This ...

Tubal Cytology of the Fallopian Tube as a Promising Tool for Ovarian Cancer Early Detection

Currently, it is widely accepted that the vast majority of ovarian high-grade serous carcinoma (HGSC) originate from the fallopian tube. However, due to the lack of markers or tools for the ovarian cancer identification, the early detection of HGSC remains challenging. Direct sampling of the fallopian tube can enhance sensitivity for detection of neoplastic cells when the tumor is not grossly visible. We developed a procedure to collect fallopian tube cells directly from freshly received surgical specimens, which has shown excellent correlation with histological findings. This approach lays a foundation for the future utility of minimally invasive laparoscopic screening in high-risk patient populations.

We explored a tubal cytologic method by sampling the fallopian tube directly post-surgical excision as a tool of ovarian cancer early detection. Here, we present a protocol to collect fallopian tube cells from freshly received surgical specimens.

Yumurtalık Kanseri Erken Teşhisi İçin Umut Verici Bir Araç Olarak Fallop Tüpünün Tubal Sitolojisi

Currently, it is widely accepted that the vast majority of ovarian high-grade serous carcinoma (HGSC) originate from the fallopian tube. However, due to ...

Accessing the Cytotoxicity and Cell Response to Biomaterials

Biomaterials contact directly or indirectly with the human tissues, making it important to evaluate its cytotoxicity. This evaluation can be performed by several methods, but a high discrepancy exists between the approaches used, compromising the reproducibility and the comparison among the obtained results. In this paper, we propose a protocol to evaluate biomaterials cytotoxicity using soluble extracts, which we use for dental biomaterials. The extracts preparation is detailed, from pellets production to its extraction in a culture medium. The biomaterials cytotoxicity evaluation is based on metabolic activity using the MTT assay, cell viability using the Sulphorhodamine B (SBR) assay, cell death profile by flow cytometry, and cell morphology using May-Gr&#252;nwald Giemsa. Additional to cytotoxicity evaluation, a protocol to evaluate cell function is described based on the expression of specific markers assessed by immunocytochemistry and PCR. This protocol provides a comprehensive guide for biomaterials cytotoxicity and cellular effects evaluation, using the extracts methodology, in a reproducible and robust manner.

This methodology aims to evaluate biomaterial cytotoxicity through the preparation of soluble extracts, using viability assays and phenotypic analysis, including flow cytometry, RT-PCR, immunocytochemistry, and other cellular and molecular biology techniques.

Sitotoksisiteye Erişim ve Biyomateryallere Hücre Yanıtı

Biomaterials contact directly or indirectly with the human tissues, making it important to evaluate its cytotoxicity. This evaluation can be performed by ...