Gli autori desiderano ringraziare James Oesterling per citometria a flusso assistenza. Questa ricerca è stata sostenuta da una sovvenzione da parte del US National Cancer Institute (R01-CA178380, ZY) e una sovvenzione US National Institutes Postdoctoral formazione (5T32CA009035-37, LWY).

L&#39;uso di animali in questo protocollo è stata effettuata in conformità con le procedure approvate dal Cancer Center Animal Care and Use Committee Fox Chase. 1 preparazione di strumenti, soluzioni e coprioggetto <ol><li> Pulire i due set di # 5 pinza sottile, un set di 7 # pinze curve sottili, una grande forbice chirurgica, una forbice microdissecting, una spatola e un cucchiaio forato. Mettere gli strumenti in un sacchetto di sterilizzazione autosigillante e autoclave. Mantenere gli strumenti in sacchetto fino al momento dell&#39;uso. </li><li> Preparare una soluzione di agarosio temperatura 3% basso punto di fusione. <ol><li> Aggiungere 1,5 g di agarosio in 50 ml di sterile Dulbecco Phosphate Buffered Saline (DPBS) poi posizionare in forno a microonde e fino alla comparsa di bolle. Pallone Swirl e riscaldare fino a quando tutti agarosio è sciolto. Mescolare la soluzione con una ancoretta prima del riscaldamento, se appaiono grumi. </li><li> Conservare la soluzione in un bagno d&#39;acqua a 37 ° C fino al momento dell&#39;uso. </li><li> Per conten-lungo terminee, conservare la soluzione di agarosio per uso futuro a temperatura ambiente oa 4 ° C per mesi. Riscaldate in forno a microonde per liquefare. Per il riscaldamento ripetuto della soluzione di agarosio, pesare il pallone prima di riscaldare per mantenere il suo peso iniziale con acqua distillata sterile tiepida. </li></ol></li><li> Preparare le seguenti soluzioni basate papaina-dissociazione cella come descritto in precedenza 2: <ol><li> Una soluzione di papaina con papaina (100 U / ml), cisteina (0,2 mg / ml) e DNasi (250 U / ml) in fenolo sterile rossi contenenti DPBS. </li><li> Una soluzione ovomucoid con albumina di siero bovino (BSA; 8 mg / ml), inibitore di tripsina di soia (8 mg / ml)) e DNasi (250 U / ml) in fenolo sterile rossi contenenti DPBS. Filtro sterilizzare entrambe le soluzioni e regolare il pH a restituire il colore originale di rosso fenolo contenenti DPBS. </li></ol></li><li> Preparare mezzi di coltura cellulare (NB / B27): integrare i media basale con 1 mM di sodio piruvato, 2 mM L-glutammina, 1% di penicillina-streptomicina (P / S) e B27 supplemento. Filter sterilizzare e proteggere dalla luce. Equilibrare i media a 37 ° C e 5% di CO 2 prima dell&#39;uso. </li><li> Preparare tampone FACS: 5% di siero fetale bovino (FBS) in DPBS. Aggiungere 2,5 ml di FBS 47,5 ml DPBS. </li><li> Preparare poli-D-lisina (PDL) coprioggetto Rivestiti. <ol><li> Autoclave nuovi vetrini. </li><li> Cappotto i coprioggetti con PDL (100 mcg / ml in acqua distillata sterile) e incubare per 2 ore a 37 ° C oa temperatura ambiente per una notte. </li><li> Lavare coprioggetto con acqua distillata sterile. Lavare una volta con NB / B27 media. Lasciate asciugare completamente coprioggetto in cappa coltura tissutale prima dell&#39;uso. </li></ol></li></ol> 2 dissezione e preparazione di fette di tessuto <ol><li> Il giorno della dissezione, pulire l&#39;area dissezione con etanolo al 70% e la copertura con un tampone assorbente. </li><li> Rimuovere strumenti di dissezione dal sacchetto di sterilizzazione. Aggiungere ghiaccio freddo, DPBS sterile / 1% P / S a una piccola scatola di Petri e posto sul ghiaccio. </li><li> Decapitare P4 Math1-GFP / Nestin-CFP cuccioli di topo con grandi forbici chirurgiche poi tenere la testa in giù con pinze curve sottili. Togliere la pelle e rimuovere delicatamente il cranio con 5 # pinza sottile con una spatola poi scavare il cervello dal cranio e trasferirlo alla piastra di Petri contenente DPBS / 1% P / S. <ol><li> Uso # 5 pinza sottile, separare accuratamente il cervelletto dal resto del cervello. Assicurati di sezionare un cucciolo alla volta il più rapidamente possibile. </li><li> Raccogliere un minimo di 8 cerebella al fine di ottenere sufficienti NEP per analisi molecolari e funzionali. </li></ol></li><li> Riempire uno stampo embedding misura 2 x 2 x 2 cm con 3% soluzione di agarosio a bassa temperatura di fusione. Assicurarsi che la temperatura di agarosio non è più di 37 ° C. </li><li> Rimuovere il liquido in eccesso dal cervelletto tamponando delicatamente su un tessuto di laboratorio. Inserire cervelletto in stampo in posizione verticale. Mettere lo stampo sul ghiaccio per consentirgli di solidificare rapidamente. Utilizzare ~ 4 cerebella per blocco. </li><li> Ottenere lisezioni di tessuto Ving con lama vibratome vibrante. Trim-cerebella blocco contenitore di agarosio con una lama di rasoio. Incollare il blocco di agarosio al piatto vibratome con cerebella orientato in posizione verticale. </li><li> Riempire il vassoio del vibratome con ghiaccio-freddo DPBS sterile / 1% P / S e posto sotto ghiaccio. <ol><li> Tagliare 600 micron sezioni attraverso tutta la lunghezza del cervelletto. Raccogliere sezioni dal vassoio di ghiaccio con un cucchiaio forato. Posizionare le sezioni in una capsula di Petri riempita con DPBS / 1% P / S sul ghiaccio. </li></ol></li></ol> 3. Microdissezione e dissociazione cellulare <ol><li> Porre la capsula di Petri contenente fette di tessuto con un microscopio a fluorescenza dissezione. <ol><li> Separare con cautela la EGL dal resto della sezione cerebellare utilizzando una pinza sottile sezionando tra lo strato PCP + molecolare e la GFP + EGL (vedi linea tratteggiata in figura 1). Fare attenzione a non includere alcuna parte del livello molecolare, che si tradurrà incontaminazione del-Nestin esprimere Bergmann glia. </li><li> Completare questo passaggio il più rapidamente possibile e mantenere il tessuto in ghiaccio per evitare la morte delle cellule. </li><li> Rimuovere l&#39;agarosio circonda il tessuto prima di procedere alla fase successiva. </li></ol></li><li> Digerire il tessuto EGL microdissezione utilizzando un protocollo basato papaina, come precedentemente descritto 2 per ottenere una sospensione di cellule singole. <ol><li> Risospendere le cellule in tampone FACS per la raccolta di cellule CFP-positivi tramite FACS. </li></ol></li></ol> 4. cellulare Ordinamento e placcatura <ol><li> Cellule Ordina per PCP fluorescenza utilizzando una sterile, flusso ad alta velocità citometro contenente un filtro PCP appropriato e raccogliere le cellule in tampone FACS su ghiaccio. Ottenere circa 100.000 NEP di ogni cervelletto P4. </li><li> Cellule centrifugare a 300 xg per 5 min. Utilizzare immediatamente le cellule per l&#39;analisi molecolare o cultura per ulteriori sperimentazioni. </li><li> Per le cellule di coltura, risospendere le cellule in pre-riscaldato NB / B27 media e piastra su vetrini PDL rivestite come precedentemente descritto 2. </li></ol>

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Il metodo microdissezione qui descritto è il primo procedimento in grado di isolare una popolazione pura di cellule viventi per uso nelle analisi molecolari e funzionali. Come dimostrato da Li et al. 9, NEP ottenuti con tale metodo può essere coltivato e incorporati in vari esperimenti e per la loro elevata purezza, possono essere utilizzati anche per analisi microarray. E &#39;molto importante prendersi il tempo per diventare esperti con questa tecnica micro...

Il cervelletto è costituito da più strati di cellule, ciascuna contenente tipi cellulari distinti. Durante lo sviluppo, la EGL contiene proliferazione dei precursori neuronali dei granuli (PNL), mentre lo strato molecolare e strato di Purkinje contengono Bergmann glia e neuroni di Purkinje, rispettivamente. Nel profondo del cervelletto si trova la sostanza bianca, che contiene le cellule staminali neurali (NSC) e oligodendrociti 1. Sonic hedgehog (Shh) svolge un ruolo critico nella regolazione dello sviluppo cerebellare e, in particolare, promuove la proliferazione di PNL tramite legame al suo recettore Patched (Ptc), un regolatore negativo di Shh segnalazione 2-4. L&#39;attivazione aberrante di segnalazione Shh genera medulloblastoma (MB), il tumore cerebrale maligno più comune nei bambini 5,6. Ptc MB mutanti modelli di topi transgenici sono strumenti potenti per lo studio di MB e si sviluppano i tumori che assomigliano MB umano 7,8. L&#39;utilizzo di questitopi, si è scoperto che PNL sono la cellula di origine per riccio di tipo MB 7. Inoltre, oltre al PNL, abbiamo recentemente identificato una popolazione unica di progenitori neuronali all&#39;interno EGL del cervelletto sviluppo che può anche dar luogo a MB. Queste cellule esprimono alti livelli di tipo VI, proteina dei filamenti intermedi, Nestin, e sono chiamati NEP 9. NEP si trovano all&#39;interno della parte profonda della EGL ed esistono transitoriamente solo durante lo sviluppo cerebellare neonatale. Essi si sono impegnati per la linea cellulare dei granuli come PNL, ma sono distinte come sono quiescenti e non esprimono Math1, un marker ben consolidata per la PNL convenzionali 10. Inoltre, NEP danno luogo a MB più efficiente rispetto PNL dopo l&#39;attivazione della via di segnalazione Shh 9, che li un romanzo origine per MB tumorigenesi fa. Abbiamo già NEP dalla cerebellare EGL isolati al giorno postnatale 4 (P4) con la tecnica microdissezionedescritto qui 9. Microdissezione tramite la visualizzazione microscopica diretta del tessuto permette di specifico dissezione del cerebellare EGL. Questo è necessario in quanto Nestin viene espressa anche dal NSC nella sostanza bianca cerebellare e da Bergmann glia nello strato molecolare 1,7,11,12 ed era fondamentale che queste cellule non essere inclusi, come avrebbero confondere l&#39;analisi. Le cellule isolate dal tessuto microdissezione possono essere utilizzati immediatamente per l&#39;analisi molecolare oppure possono essere coltivati ​​per ulteriori applicazioni. Per aiutare in modo specifico microdissecting la EGL e per purificare ulteriormente NEP, topi Math1-GFP (green fluorescent protein) sono stati incrociati con topi Nestin-CFP (proteina fluorescente ciano). Il fattore di trascrizione Math1 è specificamente espresso da PNL e l&#39;espressione della GFP è chiaramente visibile nella EGL 9,13. Topi Nestin-PCP esprimono una forma nucleare di PCP e possono essere facilmente visualizzati nello strato molecolare 9,14. Insieme, GFP e di espressione CFP creano confini per microdissezione del EGL (vedi Figura 1). NEP CFP-positivi sono stati poi isolati da FACS, dopo dissociazione enzimatica delle EGLS sezionati. Attualmente, il metodo più ben utilizzato per isolare le cellule dal EGL è Percoll centrifugazione in gradiente di tutto il tessuto cerebellare 15,16. Questo metodo, tuttavia, è in grado di escludere completamente popolazioni di cellule nestina + dal livello molecolare e la materia bianca e pertanto non può essere utilizzato per lo studio della NEP. Laser cattura microdissezione, che utilizza il trasferimento di energia laser per rimuovere le cellule di interesse, è un altro metodo usato per isolare cellule specifiche all&#39;interno di un tessuto eterogeneo 17,18 ma cellule catturate possono essere utilizzate solo per il recupero di DNA, RNA e proteine ​​e non sono in grado di essere coltivato. Questo protocollo fornisce un modo per isolare in particolare un vivo, popolazione pura della NEP dal EGL.Questa tecnica può essere applicata a diversi tipi di tessuto che hanno o architettura anatomica riconoscibile o fluorescente cellule / regioni. Pertanto, il principale vantaggio di incorporare questa nuova tecnica di microdissezione nei protocolli di isolamento delle cellule è che le cellule possono essere isolate dalle regioni tissutali specifici per eliminare la contaminazione dei tessuti vicini e le cellule possono essere raccolte immediatamente per l&#39;analisi o in coltura per altre applicazioni.

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			<td style="width:167px;">Comments/Description</td>
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			<td height="21" style="height:21px;width:215px;">Neurobasal media</td>
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			<td height="21" style="height:21px;width:215px;">PDL</td>
			<td style="width:71px;">Millipore</td>
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			<td height="42" style="height:42px;width:215px;">ultra pure low melting temperature agarose</td>
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			<td height="21" style="height:21px;width:215px;">DPBS</td>
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isolation of distinct cell populations from the developing cerebellum by microdissection

Microdissezione è una nuova tecnica in grado di isolare le regioni specifiche di un tessuto ed eliminare la contaminazione da fonti cellulari in aree adiacenti. Questo metodo è stato utilizzato nello studio di nestina-esprimendo progenitrici (NEP), una popolazione di recente identificato delle cellule dello strato germinativo cerebellare esterno (EGL). Utilizzando microdissection in combinazione con l&#39;ordinamento delle cellule fluorescenti-attivata (FACS), una popolazione pura di NEP è stato raccolto separatamente dagli convenzionali precursori neuronali dei granuli in EGL e da altri contaminanti cellule nestina-esprimono nel cervelletto. Senza microdissezione, analisi funzionali della NEP, non sarebbe stato possibile con i metodi attualmente disponibili, come Percoll centrifugazione in gradiente e microdissezione laser. Questa tecnica può essere applicata anche per l&#39;utilizzo con vari tessuti che contengono sia regioni riconoscibili o cellule fluorescenza marcata. Ancora più importante, un grande vantaggio di questo microdissectiotecnica n è che le cellule isolate vivono e possono essere coltivate per ulteriori sperimentazioni, che non è attualmente possibile con altri metodi descritti.

Come mostrato nella Figura 1A, fettine cerebellari sono stati preparati da P4 Math1-GFP / topi Nestin-PCP, in cui il cerebellare EGL è stata dominata da PNL-GFP che esprimono e lo strato molecolare è stata arricchita da cellule gliali CFP-positivi. Per isolare NEP che si trovano nella parte profonda della EGL, fette sono state microdissezionate tra EGL e lo strato molecolare (lungo la linea tratteggiata; Figura 1A). EGLS sezionato (Figura 1B) sono st...

Watch this Scientific Journal Video about Isolation of Distinct Cell Populations from the Developing Cerebellum by Microdissection at JoVE.com

Isolation of Distinct Cell Populations from the Developing Cerebellum by Microdissection

Progenitori nestina-esprimendo sono una popolazione recentemente identificato di progenitori neuronali del cervelletto in via di sviluppo. Utilizzando la tecnica di microdissezione qui presentata in combinazione con fluorescenza-attivato cell sorting, questa popolazione cellulare può essere purificato senza contaminazione da altre regioni cerebellari e può essere coltivato per ulteriori studi.

Isolamento di distinte popolazioni cellulari dal cervelletto in via di sviluppo da Microdissection

Microdissection is a novel technique that can isolate specific regions of a tissue and eliminate contamination from cellular sources in adjacent areas. ...

isolation-distinct-cell-populations-from-developing-cerebellum

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JoVE Journal

Neuroscience

9.5K Views.  Temple University Health System.  Progenitori nestina-esprimendo sono una popolazione recentemente identificato di progenitori neuronali del cervelletto in via di sviluppo. Utilizzando la tecnica di microdissezione qui presentata in combinazione con fluorescenza-attivato cell sorting, questa popolazione cellulare può essere purificato senza contaminazione da altre regioni cerebellari e può essere coltivato per ulteriori studi. 

Video: Isolation of Distinct Cell Populations from the Developing Cerebellum by Microdissection

Electrophysiological Characterization of GFP-Expressing Cell Populations in the Intact Retina

Studying the physiological properties and synaptic connections of specific neurons in the intact tissue is a challenge for those cells that lack conspicuous morphological features or show a low population density. This applies particularly to retinal amacrine cells, an exceptionally multiform class of interneurons that comprise roughly 30 subtypes in mammals1. Though being a crucial part of the visual processing by shaping the retinal output2, most of these subtypes have not been studied up to now in a functional context because encountering these cells with a recording electrode is a rare event.
Recently, a multitude of transgenic mouse lines is available that express fluorescent markers like green fluorescent protein (GFP) under the control of promoters for membrane receptors or enzymes that are specific to only a subset of neurons in a given tissue3,4. These pre-labeled cells are therefore accessible to directed microelectrode targeting under microscopic control, permitting the systematic study of their physiological properties in situ. However, excitation of fluorescent markers is accompanied by the risk of phototoxicity for the living tissue. In the retina, this approach is additionally hampered by the problem that excitation light causes appropriate stimulation of the photoreceptors, thus inflicting photopigment bleaching and transferring the retinal circuits into a light-adapted condition. These drawbacks are overcome by using infrared excitation delivered by a mode-locked laser in short pulses of the femtosecond range. Two-photon excitation provides energy sufficient for fluorophore excitation and at the same time restricts the excitation to a small tissue volume minimizing the hazards of photodamage5. Also, it leaves the retina responsive to visual stimuli since infrared light (&gt;850 nm) is only poorly absorbed by photopigments6.
In this article we demonstrate the use of a transgenic mouse retina to attain electrophysiological in situ recordings from GFP-expressing cells that are visually targeted by two-photon excitation. The retina is prepared and maintained in darkness and can be subjected to optical stimuli which are projected through the condenser of the microscope (Figure 1). Patch-clamp recording of light responses can be combined with dye filling to reveal the morphology and to check for gap junction-mediated dye coupling to neighboring cells, so that the target cell can by studied on different experimental levels.

This article depicts the recording of individual cells from fluorescently tagged neuronal populations in the intact mouse retina. By using two-photon infrared excitation transgenetically labeled cells were targeted for patch-clamp recording to study their light responses, receptive field properties, and morphology.

Caratterizzazione elettrofisiologica di GFP-Esprimendo popolazioni di cellule nella retina intatto

Studying the physiological properties and synaptic connections of specific neurons in the intact tissue is a challenge for those cells that lack ...

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Neuroscienze

Laser Capture Microdissection of Enriched Populations of Neurons or Single Neurons for Gene Expression Analysis After Traumatic Brain Injury

Long-term cognitive disability after TBI is associated with injury-induced neurodegeneration in the hippocampus-a region in the medial temporal lobe that is critical for learning, memory and executive function.1,2 Hence our studies focus on gene expression analysis of specific neuronal populations in distinct subregions of the hippocampus. The technique of laser capture microdissection (LCM), introduced in 1996 by Emmert-Buck, et al.,3 has allowed for significant advances in gene expression analysis of single cells and enriched populations of cells from heterogeneous tissues such as the mammalian brain that contains thousands of functional cell types.4 We use LCM and a well established rat model of traumatic brain injury (TBI) to investigate the molecular mechanisms that underlie the pathogenesis of TBI. Following fluid-percussion TBI, brains are removed at pre-determined times post-injury, immediately frozen on dry ice, and prepared for sectioning in a cryostat. The rat brains can be embedded in OCT and sectioned immediately, or stored several months at -80 &deg;C before sectioning for laser capture microdissection. Additionally, we use LCM to study the effects of TBI on circadian rhythms. For this, we capture neurons from the suprachiasmatic nuclei that contain the master clock of the mammalian brain. Here, we demonstrate the use of LCM to obtain single identified neurons (injured and degenerating, Fluoro-Jade-positive, or uninjured, Fluoro-Jade-negative) and enriched populations of hippocampal neurons for subsequent gene expression analysis by real time PCR and/or whole-genome microarrays. These LCM-enabled studies have revealed that the selective vulnerability of anatomically distinct regions of the rat hippocampus are reflected in the different gene expression profiles of different populations of neurons obtained by LCM from these distinct regions. The results from our single-cell studies, where we compare the transcriptional profiles of dying and adjacent surviving hippocampal neurons, suggest the existence of a cell survival rheostat that regulates cell death and survival after TBI.

We describe how to use laser capture microdissection (LCM) to obtain enriched populations of hippocampal neurons or single neurons from frozen sections of the injured rat brain for subsequent gene expression analysis using quantitative real time PCR and/or whole-genome microarrays.

Laser Microdissezione Cattura di popolazioni Arricchito di neuroni singoli neuroni o per analisi di espressione genica dopo trauma cranico

Long-term cognitive disability after TBI is associated with injury-induced neurodegeneration in the hippocampus-a region in the medial temporal lobe that ...

Production of RNA for Transcriptomic Analysis from Mouse Spinal Cord Motor Neuron Cell Bodies by Laser Capture Microdissection

Preparation of high-quality RNA from cells of interest is critical to precise and meaningful analysis of transcriptional differences among cell types or between the same cell type in health and disease or following pharmacologic treatments.&#160;In the spinal cord, such preparation from motor neurons, the target of interest in many neurologic and neurodegenerative diseases, is complicated by the fact that motor neurons represent &#60;10% of the total cell population.&#160;Laser capture microdissection (LMD) has been developed to address this problem.&#160;Here, we describe a protocol to quickly recover, freeze, and section mouse spinal cord to avoid RNA damage by endogenous and exogenous RNases, followed by staining with Azure B in 70% ethanol to identify the motor neurons while keeping endogenous RNase inhibited.&#160;LMD is then used to capture the stained neurons directly into guanidine thiocyanate lysis buffer, maintaining RNA integrity.&#160;Standard techniques are used to recover the total RNA and measure its integrity.&#160;This material can then be used for downstream analysis of the transcripts by RNA-seq and qRT-PCR.

High-quality total RNA has been prepared from cell bodies of mouse spinal cord motor neurons by laser capture microdissection after staining spinal cord sections with Azure B in 70% ethanol.&#160;Sufficient RNA (~40-60 ng) is recovered from 3,000-4,000 motor neurons to allow downstream RNA analysis by RNA-seq and qRT-PCR.

Produzione di RNA per l'analisi trascritomica dai corpi cellulari dei motoneuroni del midollo spinale del topo mediante microdisezione di cattura laser

Preparation of high-quality RNA from cells of interest is critical to precise and meaningful analysis of transcriptional differences among cell types or ...

Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the developing cerebral cortex, abnormal mitosis of neural progenitors can cause defects in brain size and function. Hence, there is a critical need for tools to understand the mechanisms of neural progenitor mitosis. Cortical development in rodents is an outstanding model for studying this process. Neural progenitor mitosis is commonly examined in fixed brain sections. This protocol will describe in detail an approach for live imaging of mitosis in ex vivo embryonic brain slices. We will describe the critical steps for this procedure, which include: brain extraction, brain embedding, vibratome sectioning of brain slices, staining and culturing of slices, and time-lapse imaging. We will then demonstrate and describe in detail how to perform post-acquisition analysis of mitosis. We include representative results from this assay using the vital dye Syto11, transgenic mice (histone H2B-EGFP and centrin-EGFP), and in utero electroporation (mCherry-&#945;-tubulin). We will discuss how this procedure can be best optimized and how it can be modified for study of genetic regulation of mitosis. Live imaging of mitosis in brain slices is a flexible approach to assess the impact of age, anatomy, and genetic perturbation in a controlled environment, and to generate a large amount of data with high temporal and spatial resolution. Hence this protocol will complement existing tools for analysis of neural progenitor mitosis.

Neural progenitor mitosis is a critical parameter of neurogenesis. Much of our understanding of neural progenitor mitosis is based on analysis of fixed tissue. Live imaging in embryonic brain slices is a versatile technique to assess mitosis with high temporal and spatial resolution in a controlled environment.

Immagini dal vivo della mitosi in via di sviluppo embrionali di topo Cortex

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the ...

Laser Capture Microdissection - A Demonstration of the Isolation of Individual Dopamine Neurons and the Entire Ventral Tegmental Area

Laser capture microdissection (LCM) is used to isolate a concentrated population of individual cells or precise anatomical regions of tissue from tissue sections on a microscope slide. When combined with immunohistochemistry, LCM can be used to isolate individual cells types based on a specific protein marker. Here, the LCM technique is described for collecting a specific population of dopamine neurons directly labeled with&#160;tyrosine hydroxylase immunohistochemistry and for isolation of the dopamine neuron containing region of the ventral tegmental area using indirect tyrosine hydroxylase immunohistochemistry on a section adjacent to those used for LCM. An infrared (IR) capture laser is used to both dissect individual neurons as well as the ventral tegmental area off glass slides and onto an LCM cap for analysis. Complete dehydration of the tissue with 100% ethanol and xylene is critical. The combination of the IR capture laser and the ultraviolet (UV) cutting laser is used to isolate individual dopamine neurons or the ventral tegmental area when using PEN membrane slides. A PEN membrane slide has significant advantages over a glass slide as it offers better consistency in capturing and collecting cells, is faster collecting large pieces of tissue, is less reliant on dehydration and results in complete removal of the tissue from the slide. Although removal of large areas of tissue from a glass slide is feasible, it is considerably more time consuming and frequently leaves some residual tissue behind. Data shown here demonstrate that RNA of sufficient quantity and quality can be obtained using these procedures for quantitative PCR measurements. Although RNA and DNA are the most commonly isolated molecules from tissue and cells collected with LCM, isolation and measurement of microRNA, protein and epigenetic changes in DNA can also benefit from the enhanced anatomical and cellular resolution obtained using LCM.

The isolation of individual dopamine neurons or the ventral tegmental area with direct or indirect immunohistochemistry is demonstrated using laser capture microdissection. Parameters for isolation of tissue from a glass slide using an infrared laser and from membrane slides using the combination of an infrared and ultraviolet laser are discussed.

Laser Capture Microdissezione - Una dimostrazione dell&#39;isolamento individuale dopamina neuroni e l&#39;intera area ventrale tegmentale

Laser capture microdissection (LCM) is used to isolate a concentrated population of individual cells or precise anatomical regions of tissue from tissue ...

In-depth Physiological Analysis of Defined Cell Populations in Acute Tissue Slices of the Mouse Vomeronasal Organ

In most mammals, the vomeronasal organ (VNO) is a chemosensory structure that detects both hetero- and conspecific social cues. Vomeronasal sensory neurons (VSNs) express a specific type of G protein-coupled receptor (GPCR) from at least three different chemoreceptor gene families allowing sensitive and specific detection of chemosensory cues. These families comprise the V1r and V2r gene families as well as the formyl peptide receptor (FPR)-related sequence (Fpr-rs) family of putative chemoreceptor genes. In order to understand the physiology of vomeronasal receptor-ligand interactions and downstream signaling, it is essential to identify the biophysical properties inherent to each specific class of VSNs.
The physiological approach described here allows identification and in-depth analysis of a defined population of sensory neurons using a transgenic mouse line (Fpr-rs3-i-Venus). The use of this protocol, however, is not restricted to this specific line and thus can easily be extended to other genetically modified lines or wild type animals.

Here, we describe a physiological approach that allows identification and in-depth analysis of a defined population of sensory neurons in acute coronal tissue slices of the mouse vomeronasal organ using whole-cell patch-clamp recordings.

Approfondita fisiologica Analisi di popolazioni di cellule definite nel acuta del tessuto Fette del mouse Vomeronasal Organo

In most mammals, the vomeronasal organ (VNO) is a chemosensory structure that detects both hetero- and conspecific social cues. Vomeronasal sensory ...

Live Imaging Followed by Single Cell Tracking to Monitor Cell Biology and the Lineage Progression of Multiple Neural Populations

Understanding the mechanisms that control critical biological events of neural cell populations, such as proliferation, differentiation, or cell fate decisions, will be crucial to design therapeutic strategies for many diseases affecting the nervous system. Current methods to track cell populations rely on their final outcomes in still images and they generally fail to provide sufficient temporal resolution to identify behavioral features in single cells. Moreover, variations in cell death, behavioral heterogeneity within a cell population, dilution, spreading, or the low efficiency of the markers used to analyze cells are all important handicaps that will lead to incomplete or incorrect read-outs of the results. Conversely, performing live imaging and single cell tracking under appropriate conditions represents a powerful tool to monitor each of these events. Here, a time-lapse video-microscopy protocol, followed by post-processing, is described to track neural populations with single cell resolution, employing specific software. The methods described enable researchers to address essential questions regarding the cell biology and lineage progression of distinct neural populations.

A robust protocol to monitor neural populations by time-lapse video-microscopy followed by software-based post-processing is described. This method represents a powerful tool to identify biological events in a selected population during live imaging experiments.

Seguita dalla singola cellula di rilevamento a Monitor biologia cellulare e la progressione di lignaggio di popolazioni neurali Multiple immagini dal vivo

Understanding the mechanisms that control critical biological events of neural cell populations, such as proliferation, differentiation, or cell fate ...

Optimization of Laser-Capture Microdissection for the Isolation of Enteric Ganglia from Fresh-Frozen Human Tissue

The purpose of this method is to obtain high-integrity RNA samples from enteric ganglia collected from unfixed, freshly-resected human intestinal tissue using laser capture microdissection (LCM). We have identified five steps in the workflow that are crucial for obtaining RNA isolates from enteric ganglia with sufficiently high quality and quantity for RNA-seq. First, when preparing intestinal tissue, each sample must have all excess liquid removed by blotting prior to flattening the serosa as much as possible across the bottom of large base molds. Samples are then quickly frozen atop a slurry of dry ice and 2-methylbutane. Second, when sectioning the tissue, it is important to position cryomolds so that intestinal sections parallel the full plane of the myenteric plexus, thereby yielding the greatest surface area of enteric ganglia per slide. Third, during LCM, polyethylene napthalate (PEN)-membrane slides offer the greatest speed and flexibility in outlining the non-uniform shapes of enteric ganglia when collecting enteric ganglia. Fourth, for distinct visualization of enteric ganglia within sections, ethanol-compatible dyes, like Cresyl Violet, offer excellent preservation of RNA integrity relative to aqueous dyes. Finally, for the extraction of RNA from captured ganglia, we observed differences between commercial RNA extraction kits that yielded superior RNA quantity and quality, while eliminating DNA contamination. Optimization of these factors in the current protocol greatly accelerates the workflow and yields enteric ganglia samples with exceptional RNA quality and quantity.

The goal of this protocol is to obtain high-integrity RNA samples from enteric ganglia isolated from unfixed, freshly-resected human intestinal tissue using laser capture microdissection (LCM). This protocol involves preparing flash-frozen samples of human intestinal tissue, cryosectioning, ethanolic staining and dehydration, LCM, and RNA extraction.

Ottimizzazione di Laser Capture Microdissection per l'isolamento dei gangli enterici dal tessuto umano fresco congelato

The purpose of this method is to obtain high-integrity RNA samples from enteric ganglia collected from unfixed, freshly-resected human intestinal tissue ...

A Stainless Protocol for High Quality RNA Isolation from Laser Capture Microdissected Purkinje Cells in the Human Post-Mortem Cerebellum

Laser capture microdissection (LCM) is an advantageous tool that allows for the collection of cytologically and/or phenotypically relevant cells or regions from heterogenous tissues. Captured product can be used in a variety of molecular methods for protein, DNA or RNA isolation. However, preservation of RNA from postmortem human brain tissue is especially challenging. Standard visualization techniques for LCM require histologic or immunohistochemical staining procedures that can further degrade RNA. Therefore, we designed a stainless protocol for visualization in LCM with the intended purpose of preserving RNA integrity in post-mortem human brain tissue. The Purkinje cell of the cerebellum is a good candidate for stainless visualization, due to its size and characteristic location. The cerebellar cortex has distinct layers that differ in cell density, making them a good archetype to identify under high magnification microscopy. Purkinje cells are large neurons situated between the granule cell layer, which is a densely cellular network of small neurons, and the molecular layer, which is sparse in cell bodies. Because of this architecture, the use of stainless visualization is feasible. Other organ or cell systems that mimic this phenotype would also be suitable. The stainless protocol is designed to fix fresh-frozen tissue with ethanol and remove lipids with xylene for improved morphological visualization under high magnification light microscopy. This protocol does not account for other fixation methods and is specifically designed for fresh-frozen tissue samples captured using an ultraviolet (UV)-LCM system. Here, we present a full protocol for sectioning and fixing fresh frozen post-mortem human cerebellar tissue and purification of RNA from Purkinje cells isolated by UV-LCM, while preserving RNA quality for subsequent RNA-sequencing. In our hands, this protocol produces exceptional levels of cellular visualization without the need for staining reagents and yields RNA with high RNA integrity numbers (&#8805;8) as needed for transcriptional profiling experiments.

This protocol uses a stain-free approach to visualize and isolate Purkinje cells in fresh-frozen tissue from human post-mortem cerebellum via laser capture microdissection. The purpose of this protocol is to generate sufficient amounts of high-quality RNA for RNA-sequencing.

Un protocollo di acciaio inox per l'isolamento di RNA di alta qualità da Laser catturare cellule Microdissected Purkinje nel cervelletto umano Post-Mortem

Laser capture microdissection (LCM) is an advantageous tool that allows for the collection of cytologically and/or phenotypically relevant cells or ...

Primary Culture of Neurons Isolated from Embryonic Mouse Cerebellum

The use of primary cell cultures has become one of the major tools to study the nervous system in vitro. The ultimate goal of using this simplified model system is to provide a controlled microenvironment and maintain the high survival rate and the natural features of dissociated neuronal and nonneuronal cells as much as possible under in vitro conditions. In this article, we demonstrate a method of isolating primary neurons from the developing mouse cerebellum, placing them in an in vitro environment, establishing their growth, and monitoring their viability and differentiation for several weeks. This method is applicable to embryonic neurons dissociated from cerebellum between embryonic days 12&ndash;18.

Conducting in vitro experiments to reflect in vivo conditions as adequately as possible is not an easy task. The use of primary cell cultures is an important step toward understanding cell biology in a whole organism. The provided protocol outlines how to successfully grow and culture embryonic mouse cerebellar neurons.

Cultura primaria dei neuroni isolata dal cerbellello del topo embrionale

The use of primary cell cultures has become one of the major tools to study the nervous system in vitro. The ultimate goal of using this simplified model ...