Name: visualization of the interstitial cells of cajal (icc) network in mice
Uploaded: 2011-07-27T12:00:00
Duration: 9 min 45 s
Description: Watch this Scientific Journal Video about Visualization of the Interstitial Cells of Cajal ICC Network in Mice at JoVE.com

This work was supported by the National Cancer Institute (K08CA140946, to Y.C.), (5F32CA130372, to P.C.), (R01CA102774 and R01HL055748 to P.B.), the Department of Defense (PC094302 to Y.C.), the Shuman Family Fund for GIST Research (to P.C.), and the Starr Cancer Consortium (to P.C., Y.C., C.L.S. and P.B.). We would like
to thank Katia Manova, Ning Fan, and Mesurh Turkekul of the MSKCC
Molecular Cytology core facility for help with cryosectioning and
immunostaining.

1. Dissection of mouse GI tract
<ol>
<li>Euthanized mice by local IUCAC guidelines</li> 
<li>Pin each limb on Styrofoam surface and rinse abdominal surface with 70% ethanol. Open abdominal cavity with long midline incision from the diaphragm to pubis. Make one cut at the distal esophagus and a second cut at the distal large intestine and remove the GI tract from stomach to anus en bloc, carefully cutting ligaments at the duodenum and cecum using small scissors. Place GI tract in a petri dish with PBS. Dissect away mesentery using tweezers (Figure 1). To separate stomach, small intestine, and large intestine, cut at pylorus and ileocecal junction. Rinse the lumen of each part of the GI tract with PBS using a 5ml syringe attached to a feeding needle (older mice) or blunt needle (younger mice).</li>
<li>Open stomach, small intestine, and large intestine along the mesenteric border. Cut two pieces for each part of the GI tract, one for whole mount visualization and one for cryosection.</li>
</ol>
2. Whole mount sample preparation and immunostaining
<ol>
<li>Place the entire piece of each part of GI tract into a 1.5 cm microfuge tube filled with 1.0 ml of ice cold acetone and fix samples for at least 1 hour at 4&deg;C. Samples may be stored for up to 1 week at -20 &deg;C. We typically store samples in acetone and proceed to prepare cyrosections.</li>
<li>Wash sample 2X with PBS. Place sample in petri dish with PBS. Under dissecting miscroscope, carefully scrape off mucosal layer with a scalpel by holding one end with a pair of tweezers, leaving the muscularis.</li>
<li>Muscle can be cut into 5mm pieces for staining with different antibodies. Do not store for more than 2 days in PBS before staining.</li>
<li>Block and incubation steps can be done in one well of a 24-well plate or in a 1.5ml microfuge tube. Block with 0.5 ml of blocking buffer (5% goat serum, 0.1% Triton X-100 in PBS for 1 hour at 4 &deg;C).</li>
<li>Incubate with primary antibody diluted in blocking buffer and rotate overnight at 4 &deg;C. We have successfully used ACK-2 rat anti-Kit, rabbit anti-Ano1, and rabbit anti-Pgp9.5 antibodies for whole mount immunostaining.</li> 
<li>Wash for 5 min 2X with PBS on rotator at room temperature.</li> 
<li>Incubate with secondary antibody diluted in blocking buffer for 2 hours at room temperature on rotator. We typically use Alexa Fluor 488 anti-rabbit and Alexa Fluor 594 anti-rat antibodies.</li>
<li>Wash for 15 min 3X with PBS on rotator at room temperature.</li>
<li>Mount on slide, serosal side on top to ensure the orientation of tissue during microscopy. This orientation will ensure the serosal side to face the coverslip and be encountered first as one focuses into the slide on a microscope. We do this on a dissecting microscope. Sometimes, we briefly examine the tissue under fluorescence microscopy to properly orient. Under fluorescence microscopy to visualize Kit or Ano1, one should encounter the ICC-MY network first from the side of the coverslip. Next, aspirate PBS as much as possible without completely drying. Add 50 &mu;l of hard set mounting media (we use Prolong Gold) and gently place cover slip on top of tissue. Allow setting of mounting overnight at room temperature and store slides at -80 &deg;C.</li>
<li>For microscopic examination, we used a wide-field microscope with a Z-drive. We used either a 20X air objective (NA 0.75, plan apochromat) or 60X oil objective (NA 1.4, plan apochromat) and took 2 &mu;m or 1 &mu;m Z-sections that span the entire muscularis. The images were subsequently deconvoluted using Autoquant deconvolution software. Alternatively, many others have used confocal microscopy instead with similar results.</li>
</ol>
3. Cryosection preparation and immunostaining
<ol>
<li>Place each part of the GI tract, serosal side down, flat on filter paper. Cut filter paper around the tissue. Place tissue on filter paper in 4% paraformaldehyde (PFA) in PBS (we dilute 32% paraformaldehyde in sealed vial into PBS right before use) for 2 hours at room temperature. Fixation can be done in a 6- or 12-well plate depending on size of tissue, or in a 1.5 ml microfuge tube. If done in plate, parafilm to minimize PFA exposure. Longer fixation times may result in antigen masking, particularly for the ACK-2 anti-Kit antibody.</li>
<li>Remove tissue from PFA and place in 30% sucrose in PBS. Allow tissue to sink overnight in 4 &deg;C.</li>
<li>Equilibrate tissue with 1:1 ratio of 30% sucrose and O.C.T. for 1 hour. Then mount tissue vertically in cryomold filled with O.C.T. The longitudinal axis of the GI tract should be in parallel with the cyrosections. When examining mice with different genotypes or with different treatments, it may be useful to mount tissue from different mice onto same cryomold to ensure parallel processing. Freeze on dry ice and place in -80 &deg;C freezer for storage.</li>
<li>Cut 10 &mu;m cryosections onto slide and store at -80 &deg;C freezer. We usually cut two sections per slide. We also usually perform H&amp;E staining of one cryosection slide.</li>
<li>For immunofluorescence, first equilibrate slide at room temperature for 15 minutes. Then draw circle around tissue using PAP pen.</li> 
<li>Block with 150 &mu;l of blocking buffer (same as for whole mount immunostaining) for 1 hour.</li>
<li>Aspirate blocking buffer and add 150 &mu;l primary antibody diluted in blocking buffer and incubate overnight at 4 &deg;C in a humidified chamber.</li>
<li>Wash 5 min 2X with PBS.</li>
<li>Incubate with secondary antibody diluted in blocking buffer for 2 hours at room temperature. We typically use Alexa 488 anti-rabbit and Alexa 594 anti-rat antibodies.</li>
<li>Wash for 15 min 3X with PBS.</li>
<li>Aspirate slide without completely drying tissue. Add a drop of hard set mounting media with DAPI (we use Prolong Gold) and place #1.5 coverslip. Place slide at room temperature overnight to set and store in -80 &deg;C freezer.</li>
<li>Acquire triple fluorescence images using DAPI, FITC, and Texas Red filter sets.</li>
</ol>
4. Representative Results: 
Here, we use the large intestine as an example. H&amp;E stain of a sucrose protected fixed frozen section used for immunofluorescence shows some artifacts compared to a reference paraffin section (Figure 2), where the yellow line demarks the myenteric plexus between the circular and longitudinal muscle layers and the green line demarks the border between the mucosa and muscularlis. Examining the cryosection, from the serosa to mucosa, you first encounter the thinner longitudinal muscle (LM) layer followed by the thicker circular muscle (CM) layer. The section should be in parallel with the longitudinal muscle so the nuclei of LM should be somewhat elongated while the nuclei of the CM should be small and round. Between the LM and CM are myenteric plexi made of neurons that stain with Pgp9.5. Pgp9.5 also stains neuronal processes throughout the CM. The myenteric ICC network (ICC-MY) surround the myenteric plexus and are made of multipolar cells. Within the longitudinal muscle layer, there are rare intramuscular ICCs (ICC-IMs) which are bipolar cells running in parallel with the muscle. Within the circular muscle layer, there are abundant ICC-IMs which are also bipolar cells that run parallel with the circular muscle and are cross-sectioned with this cut. At the junction of the muscularis and submucosa, the submucosal ICC network consisting of ICC-SMPs is a flat network of multipolar ICCs. Examining the whole mount Kit immunostaining of the large intestine, one should expect to encounter the ICC-MY network first, and then the ICC-IM of the CM, and ICC-SMP networks, as one focuses from the coverslip (Figure. 3). Volumetric measure of ICC networks using 3D reconstruction of whole-mount images have been described10. Using wide-field microscope, there is significant out-of-plane fluorescence in the raw images which can be removed after deconvolution (Figure 3). Incomplete stripping of mucosa will result in high background fluorescence. ANO1 is another marker of ICC&#x2019;s and co-immunostaining of KIT and ANO1 shows complete overlap of ICC staining (Figure 4).
<img src="/files/ftp_upload/2802/2802fig1.jpg" alt="Figure 1"/> 
Figure 1. Representative dissected mouse gastrointestinal tract, reprinted with permission13.
<img src="/files/ftp_upload/2802/2802fig2.jpg" alt="Figure 2"/> 
Figure 2. A) Representative H&amp;E staining of the mouse large intestine of cryosections prepared as described and reference paraffin embedded section showing some shrinkage artifacts of cryosections. The yellow line demarks the myenteric plexus and the blue line separates the mucosa from the muscularis. B) Representative Kit (red, ACK-2 ) and Pgp9.5 (green) double immunofluorescence with DAPI (blue) counterstaining of the mouse large intestine. LM: longitudinal muscle CM: circular muscle. Scale bar 20 &mu;m.
<img src="/files/ftp_upload/2802/2802fig3.jpg" alt="Figure 3"/> 
Figure 3. Representative Kit (red, ACK-2) whole-mount immunofluorescence of the same field at three focal planes, the ICC-MY plane at the LM/CM border, the ICC-IM plane in the CM, and the ICC-SMP plane at the CM/submucosa border. Raw images and deconvoluted images are shown. Scale bar 20 &mu;m.
<img src="/files/ftp_upload/2802/2802fig4.jpg" alt="Figure 4"/> 
Figure 4. Representative double-immunofluorescence of Ano1 (green) and Kit (red, ACK-2) in large intestine. Scale bar 20 &mu;m.

<ol>
	<li>Sanders, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+case+for+interstitial+cells+of+Cajal+as+pacemakers+and+mediators+of+neurotransmission+in+the+gastrointestinal+tract.">A case for interstitial cells of Cajal as pacemakers and mediators of neurotransmission in the gastrointestinal tract.</a> Gastroenterology. 111, 492-515 (1996).</li><li>Ward, S. M., Sanders, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiology+and+pathophysiology+of+the+interstitial+cell+of+Cajal:+from+bench+to+bedside.+I.+Functional+development+and+plasticity+of+interstitial+cells+of+Cajal+networks.">Physiology and pathophysiology of the interstitial cell of Cajal: from bench to bedside. I. Functional development and plasticity of interstitial cells of Cajal networks.</a> Am J Physiol Gastrointest Liver Physiol. 281, 602-611 (2001).</li><li>Komuro, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+organization+of+interstitial+cells+of+Cajal+in+the+gastrointestinal+tract.">Structure and organization of interstitial cells of Cajal in the gastrointestinal tract.</a> J Physiol. 576, 653-658 (2006).</li><li>Sanders, K. M., Ordog, T., Koh, S. D., Torihashi, S., Ward, 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=Development+and+plasticity+of+interstitial+cells+of+Cajal.">Development and plasticity of interstitial cells of Cajal.</a> Neurogastroenterol Motil. 11, 311-338 (1999).</li><li>Ward, S. M., Burns, A. J., Torihashi, S., Sanders, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutation+of+the+proto-oncogene+c-kit+blocks+development+of+interstitial+cells+and+electrical+rhythmicity+in+murine+intestine.">Mutation of the proto-oncogene c-kit blocks development of interstitial cells and electrical rhythmicity in murine intestine.</a> J Physiol. 480, 91-97 (1994).</li><li>Huizinga, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=W/kit+gene+required+for+interstitial+cells+of+Cajal+and+for+intestinal+pacemaker+activity.">W/kit gene required for interstitial cells of Cajal and for intestinal pacemaker activity.</a> Nature. 373, 347-349 (1995).</li><li>Rubin, B. P., Heinrich, M. C., Corless, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gastrointestinal+stromal+tumour.">Gastrointestinal stromal tumour.</a> Lancet. 369, 1731-1741 (2007).</li><li>Hirota, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gain-of-function+mutations+of+c-kit+in+human+gastrointestinal+stromal+tumors.">Gain-of-function mutations of c-kit in human gastrointestinal stromal tumors.</a> Science. 279, 577-580 (1998).</li><li>Chi, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ETV1+is+a+lineage-specific+survival+factor+in+Gastrointestinal+Stromal+Tumour+(GIST).">ETV1 is a lineage-specific survival factor in Gastrointestinal Stromal Tumour (GIST).</a> Nature. , (2010).</li><li>Kwon, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+the+structure+and+function+of+ICC+networks+in+ICC+hyperplasia+and+gastrointestinal+stromal+tumors.">Changes in the structure and function of ICC networks in ICC hyperplasia and gastrointestinal stromal tumors.</a> Gastroenterology. 136, 630-639 (2009).</li><li>Hwang, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+anoctamin+1/TMEM16A+by+interstitial+cells+of+Cajal+is+fundamental+for+slow+wave+activity+in+gastrointestinal+muscles.">Expression of anoctamin 1/TMEM16A by interstitial cells of Cajal is fundamental for slow wave activity in gastrointestinal muscles.</a> J Physiol. 587, 4887-4904 (2009).</li><li>Gomez-Pinilla, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ano1+is+a+selective+marker+of+interstitial+cells+of+Cajal+in+the+human+and+mouse+gastrointestinal+tract.">Ano1 is a selective marker of interstitial cells of Cajal in the human and mouse gastrointestinal tract.</a> Am J Physiol Gastrointest Liver Physiol. 296, G1370-G1381 (2009).</li><li>Sommer, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gastrointestinal+stromal+tumors+in+a+mouse+model+by+targeted+mutation+of+the+Kit+receptor+tyrosine+kinase.">Gastrointestinal stromal tumors in a mouse model by targeted mutation of the Kit receptor tyrosine kinase.</a> Proc Natl Acad Sci U S A. 100, 6706-6711 (2003).</li></ol>

No conflicts of interest declared.

Interstitial cells of Cajal were initially characterized by Santiago Ram&oacute;&oacute;n y Cajal exactly one century ago using methylene blue and silver chromate stains of gastrointestinal muscularis. Cajal initially thought ICC&#x2019;s were neurons based on their processes that are reminiscent of axons and dendrites. Over many ensuing years, study of ICC biology has been limited by lack of specific markers until the discovery of that KIT not only is expressed in ICC, but is also required for their development6. Since then, KIT immunofluorescence has been widely used in the study of ICC biology and has also led to the appreciation of ICC in other contractile organs such as the urinary bladder. Recently, ANO1 has been identified as a second reliable ICC marker.
There have been numerous publications over the past 15 years using immunofluorescence to identify ICC using various fixation and mounting techniques. In our hands "fixed-frozen" cryosections that have been prefixed with paraformaldehyde and sucrose protected works best compared to acetone or paraformaldehyde post-fixation after cryosectioning. For whole mounts, we have found that acetone fixation followed by mucosal dissection gives the most robust results.
Historically, the ACK-2 has been the Kit antibody of choice for mouse ICC identification. It is a rat monoclonal that recognizes the extracellular domain and it is also a blocking antibody that causes ileus when given to live mice. However, the ACK-2 epitope is slowly destroyed by paraformaldehyde fixation and is not rescued by standard antigen retrieval methods. We have found that the ACK-4 epitope is more resistant to paraformaldehyde fixation. Compared to fixed frozen blocks and sections, paraffin embedded blocks and sections preserves morphology better and is more amenable to long term archiving (Figure 4). Neither ACK-2 nor ACK-4 have been successfully used in paraffin sections. We have characterized that D13A2, a new rabbit monoclonal antibody, works exceptionally well in both fixed-frozen and paraffin sections of the GI tract.
Kit is expressed in several other cell types, including melanocytes, hematopoietic cells, germ cells, and in particular mast cells which are also found in the GI tract. Luckily, most mast cells are found is the mucosa and not in the muscularis. Double staining with rabbit anti-Ano1 and rat anti-Kit can eliminate non-specific staining of each antibody (Figure 4). It is also worth noting that the relative expression of Kit and Ano1 between subclasses of ICC seems different. For example, in the small intestine, Kit staining is much more intense in myenteric ICC compared to ICC of the deep muscle plexus whereas Ano1 staining is comparable.

<table border="1">
	<tbody>
 	<tr>
 	<th>Name of the reagent</th>
 <th>Company</th>
 <th>Catalogue number</th>
 <th>Comments (optional)</th>
 </tr>
 <tr>
 	<td>rat anti-Kit antibody (clone ACK-2)</td>
 <td>eBioscience</td>
 <td>14-1172</td>
 <td>Use at 2&mu;g/ml. Epitope is lost with overfixation.</td>
 </tr>
 <tr>
 	<td>rat anti-Kit antibody (clone ACK-4)</td>
 <td>Cedarlane</td>
 <td>CL8936AP</td>
 <td>Use at 2&mu;g/ml.</td>
 </tr>
 <tr>
 	<td>rabbit anti-KIT antibody (clone D13A2)</td>
 <td>Cell Signaling</td>
 <td>3074</td>
 <td>Use at 1:100 dilution. Only antibody listed here that works well in paraffin sections.</td>
 </tr>
 <tr>
 	<td>rabbit anti-Pgp9.5 antibody</td>
 <td>Abcam</td>
 <td>ab10404</td>
 <td>Labels neuronal cell cytoplasm. Useful for marking neurons of the myenteric plexus. Use at 1:1000 dilution.</td>
 </tr>
 <tr>
 	<td>rabbit anti-Ano1 antibody</td>
 <td>Abcam</td>
 <td>ab53212</td>
 <td>Use at 2&mu;g/ml</td>
 </tr>
 <tr>
 	<td>Alexa Fluor 594 goat anti-rat IgG</td>
 <td>Invitrogen</td>
 <td>A-11007</td>
 <td>Use at 2&mu;g/ml</td>
 </tr>
 <tr>
 	<td>Alexa Fluor 488 goat anti-rabbit IgG </td>
 <td>Invitrogen</td>
 <td>A-11008</td>
 <td>Use at 2&mu;g/ml</td>
 </tr>
 <tr>
 	<td>Animal Feeding Needle</td>
 <td>Fisher</td>
 <td>01-208-87</td>
 <td>Silicon tipped needle useful for flushing GI tract of adult mice</td>
 </tr>
 <tr>
 	<td>Blunt Needle</td>
 <td>Becton Dickison</td>
 <td>305180</td>
 <td>Blunt needle useful for flushing GI tract of pre-weaned pups</td>
 </tr>
 <tr>
 	<td>ProLong Gold antifade reagent with DAPI</td>
 <td>Invitrogen</td>
 <td>P-36931</td>
 <td>May use alternative hard-setting mounting media with DAPI</td>
 </tr>
 <tr>
 	<td>Tissue-Tek Cryomold</td>
 <td>Tissue-Tek</td>
 <td>4557</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>Tissue-Tek O.C.T.</td>
 <td>Tissue-Tek</td>
 <td>4583</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>32% Paraformaldehyde (10ml sealed ampoule) </td>
 <td>EM Sciences</td>
 <td>15714</td>
 <td>Open sealed ampoule and dilute to 4% just before use</td>
 </tr>
 <tr>
 	<td>Blocking buffer</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>5% Goat serum, 0.1% Triton X-100 in PBS</td>
 </tr> 
 </tbody>
</table>

visualization of the interstitial cells of cajal (icc) network in mice

The interstitial cells of Cajal (ICC) are mesenchymal derived "pacemaker cells" of the gastrointestinal (GI) tract that generate spontaneous slow waves required for peristalsis and mediate neuronal input from the enteric nervous system1. Different subtypes of ICC form distinct networks in the muscularis of the GI tract 2,3. Loss or injury to these networks is associated with a number of motility disorders4. ICC cells express the KIT receptor tyrosine kinase on the plasma membrane and KIT immunostaining has been used for the past 15 years to label the ICC network5,6. Importantly, normal KIT activity is required for ICC development5,6. Neoplastic transformation of ICC cells results in gastrointestinal stromal tumor (GIST), that frequently harbor gain-of-function KIT mutations7,8. We recently showed that ETV1 is a lineage-specific survival factor expressed in the ICC/GIST lineage and is a master transcriptional regulator required for both normal ICC network formation and for of GIST tumorigenesis9. We further demonstrate that it cooperates with activating KIT mutations in tumorigenesis. Here, we describe methods for visualization of ICC networks in mice, largely based on previously published protocols10,11. More recently, the chloride channel anoctamin 1 (ANO1) has also been characterized as a specific membrane marker of ICC11,12. Because of their plasma membrane localization, immunofluorescence of both proteins can be used to visualize the ICC networks. Here, we describe visualization of the ICC networks by fixed-frozen cyrosections and whole mount preparations.

Watch this Scientific Journal Video about Visualization of the Interstitial Cells of Cajal ICC Network in Mice at JoVE.com

Visualization of the Interstitial Cells of Cajal ICC Network in Mice

The interstitial cells of Cajal (ICC) are the pacemaker cells of the gastrointestinal (GI) tract. They form complex networks between smooth muscle cells and post-ganglionic neuronal fibers to regulate GI contractility. Here, we present immunofluorescence methods cross-sectional and whole-mount visualization of murine ICC networks.

Visualization of the Interstitial Cells of Cajal (ICC) Network in Mice

The interstitial cells of Cajal (ICC) are mesenchymal derived "pacemaker cells" of the gastrointestinal (GI) tract that generate spontaneous slow waves ...

visualization-of-the-interstitial-cells-of-cajal-icc-network-in-mice

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Biology

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A rapid technique for the visualization of growing immobilized yeast cells, here applied to fluorescent reporters at the silent mating loci HML and HMR

We present here a simple, rapid, and extremely flexible technique for the immobilization and visualization of growing yeast cells by epifluorescence ...

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Determining the number of cells in culture is important in standardization of culture conditions and in performing accurate quantitation experiments. In this video, we demonstrate how cells are counted using a hemacytometer.

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Cultured mammalian cells are used extensively in cell biology studies. This video describes the basic skills required to freeze and store cells and how to recover frozen stocks.

Cultured mammalian cells are used extensively in cell biology studies. It requires a number of special skills in order to be able to preserve the ...

Microarray Analysis for Saccharomyces cerevisiae

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen peroxide (H2O2), an oxidizing agent. In the experiment, yeast is grown for 48 hours in 1/2X YPD broth containing 3X glucose. The culture is split into a control and treated group. The experiment culture is treated with 0.5 mM H2O2 in Hanks Buffered Saline (HBSS) for 1 hour. The control culture is treated with HBSS only. Total RNA is extracted from both cultures and is converted to a biotin-labeled cRNA product through a multistep process. The final synthesis product is taken back to the UVM Microarray Core Facility and hybridized to the Affymetrix yeast GeneChips. The resulting gene expression data are uploaded into bioinformatics data analysis software.

Microarray Analysis for Saccharomyces cerevisiae

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen peroxide (H2O2), an oxidizing agent.

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen ...

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The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

Visualization of Endoplasmic Reticulum Localized mRNAs in Mammalian Cells

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum (ER). However, the visualization of these mRNAs can be challenging. This is especially true when only a fraction of the mRNA is ER-associated and their distribution to this organelle is obstructed by non-targeted (i.e. "free") transcripts. In order to monitor ER-associated mRNAs, we have developed a method in which cells are treated with a short exposure to a digitonin extraction solution that selectively permeabilizes the plasma membrane, and thus removes the cytoplasmic contents, while simultaneously maintaining the integrity of the ER. When this method is coupled with fluorescent in situ hybridization (FISH), one can clearly visualize ER-bound mRNAs by fluorescent microscopy. Using this protocol the degree of ER-association for either bulk poly(A) transcripts or specific mRNAs can be assessed and even quantified. In the process, one can use this assay to investigate the nature of mRNA-ER interactions.

Here we describe a method to visualize endoplasmic reticulum-associated mRNAs in mammalian tissue culture cells. This technique involves the selective permeabilization of the plasma membrane with digitonin to remove cytoplasmic contents followed by fluorescent in situ hybridization to detect either bulk poly(A) mRNA or specific transcripts.

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

Visualization of Endoplasmic Reticulum Subdomains in Cultured Cells

The lipids and proteins in eukaryotic cells are continuously exchanged between cell compartments, although these retain their distinctive composition and functions despite the intense interorganelle molecular traffic. The techniques described in this paper are powerful means of studying protein and lipid mobility and trafficking in vivo and in their physiological environment. Fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) are widely used live-cell imaging techniques for studying intracellular trafficking through the exo-endocytic pathway, the continuity between organelles or subcompartments, the formation of protein complexes, and protein localization in lipid microdomains, all of which can be observed under physiological and pathological conditions. The limitations of these approaches are mainly due to the use of fluorescent fusion proteins, and their potential drawbacks include artifactual over-expression in cells and the possibility of differences in the folding and localization of tagged and native proteins. Finally, as the limit of resolution of optical microscopy (about 200 nm) does not allow investigation of the fine structure of the ER or the specific subcompartments that can originate in cells under stress (i.e. hypoxia, drug administration, the over-expression of transmembrane ER resident proteins) or under pathological conditions, we combine live-cell imaging of cultured transfected cells with ultrastructural analyses based on transmission electron microscopy.

We describe the imaging approaches we use to investigate the distribution and mobility of the transfected fluorescent proteins resident in the endoplasmic reticulum (ER) by means of the confocal imaging of living cells. We also ultrastructurally analyze the effect of their expression on the architecture of this subcellular compartment.

The lipids and proteins in eukaryotic cells are continuously exchanged between cell compartments, although these retain their distinctive composition and ...

Visualization of G3BP Stress Granules Dynamics in Live Primary Cells

SGs can be visualized in cells by immunostaining of specific protein components or polyA+ mRNAs. SGs are highly dynamic and the study of their assembly and fate is important to understand the cellular response to stress. The deficiency in key factors of SGs like G3BP (RasGAP SH3 domain Binding Protein) leads to developmental defects in mice&#160;and alterations of the Central Nervous System. To study the dynamics of SGs in cells from an organism, one can culture primary cells and follow the localization of a transfected tagged component of SGs. We describe time-lapse experiment to observe G3BP1-containing SGs in Mouse Embryonic Fibroblasts (MEFs). This technique can also be used to study G3BP-containing SGs in live neurons, which is crucial as it was recently shown that these SGs are formed at the onset of neurodegenerative diseases like Alzheimer&#39;s disease. This approach can be adapted to any other cellular body and granule protein component, and performed with transgenic animals, allowing the live study of granules dynamics for example in the absence of a specific factor of these granules.

Stress granules (SGs) are cytoplasmic RNA granules containing stalled ribonucleoprotein particles (RNPs), and important in cellular response to various stresses. Dynamics of SGs can be followed in live cells by visualizing the localization of a tagged component of SGs in transfected primary cells after stress.

SGs can be visualized in cells by immunostaining of specific protein components or polyA+ mRNAs. SGs are highly dynamic and the study of their assembly ...