This research was supported by Grants-in-Aid for Young Scientists (A) (#16680015), Grant-in-Aid for Exploratory Research (#16653065), Grant-in-Aid for Scientific Research on Priority Areas (#18023022, #18016012 and #18053015) from the Ministry of Education, Culture, Sports, Science, and Technology in Japan, Grant-in-Aid from Promotion of Fundamental Studies in Health Sciences of the National Institute of Biomedical Innovation (NIBIO), Grant-in-Aid from Neuroinformatics Japan Center (NIJC), RIKEN

<ol><li>The apparatus used for the light/dark transition test consisted of a cage (21x42x25 cm) divided into two sections of equal size by a partition with door (Ohara &amp; Co., Tokyo).</li><li>Mice are housed three to four per cage in a room with a 12 hr light/dark cycle (lights on at 7:00 A.M.) with ad libitum access to food and water. Ideally, two controls and two mutants are housed together, by re-organizing the mice as soon as they are genotyped at the time of weaning. Behavioral testing is performed between 9:00 A.M. and 6:00 P.M. All the cages containing mice are transferred to the behavior testing room 30 min before the first trial begins.</li><li>One chamber is brightly illuminated by white diodes (390 lux), whereas the other chamber is dark (2 lux). Mice are placed into the dark side and the door is opened automatically 3 seconds after the mouse is detected by the infrared camera. The door is used so that the mice do not enter the light chamber immediately after the release with their motivation to escape from experimenter, since the latency to enter the light chamber may serve as an index of anxiety-like behavior. </li><li>Mice are allowed to move freely between the two chambers with door open for 10 min. The application used for acquiring and analyzing the behavioral data (Image LD4) is based on the public domain Image J program (developed byWayne Rasband at the National Institute of Mental Health and available at http://rsb.info.nih.gov/ij/), which was modified by Tsuyoshi Miyakawa (available through O'Hara &amp; Co., Tokyo, Japan). </li><li>The distance traveled in each chamber, the total number of transitions, the time spent in the each chamber, and the latency to enter the light chamberare recorded by Image LD4 program. </li><li>After each trial, all chambers are cleaned with super hypochlorous water to prevent a bias based on olfactory cues.</li></ol>

<ol>
	<li>Austin, C. P., Aiba, A., Takao, K., Miyakawa, T., Takao, K., Yamasaki, N., Miyakawa, T., Crawley, J. N., Crawley, J. N., Crawley, J., Goodwin, F. K., Miyakawa, T., Crabbe, J. C., Wahlsten, D., Dudek, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+knockout+mouse+project.">The knockout mouse project.</a> Nat Genet. 36, 921-924 (2004).</li></ol>

All procedures were approved by the Animal Use and Care Committee of Kyoto University.
Tsuyoshi Miyakawa is associated with BIRD, Japan Science and Technology Agency, Saitama, Japan.

Although all of the mouse genome sequences have been determined, the functions of most of these genes are not known. Gene-targeting techniques, however, can be used to delete or manipulate a specific gene in mice(Aiba et al., 2007; Austin et al., 2004). The influence of a given gene on a specific behavior can then be determined by conducting behavioral analyses of the mutant mice(Takao and Miyakawa, 2006; Takao et al., 2007). As a test for behavioral phenotyping of mutant mice, the light/dark transition test is one of the most widely used tests to measure anxiety-like behavior in mice(Crawley, 2000). The test is based on the natural aversion of mice to brightly illuminated areas and on their spontaneous exploratory behavior in novel environments(Crawley, 1985). The test is sensitive to anxiolytic drug treatment(Crawley, 1985). The apparatus consists of a dark chamber and a brightly illuminated chamber. A restricted opening, 3 cm high by 5 cm wide, connects the two chambers. Mice are allowed to move freely between the two chambers. The number of entries into the bright chamber and the duration of time spent there are used as indices of bright-space anxiety in mice.The light/dark transition test was originally developed by Crawley and colleagues (Crawley and Goodwin, 1980). There are two differences between their original version and our test. First, the light chamber is larger than the dark chamber in the original version, whereas the size of the two chambers is the same in our version of the test. Second, in the original version, the light chamber had no ceiling and the walls of the light chamber were transparent(Crawley and Goodwin, 1980), while we use opaque white plastic for the ceiling and walls of the light chamber. These differences, namely the size and openness of the light chamber, allow for the simultaneous detection of bright-space anxiety as well as open-space anxiety in the original version of the test. In our laboratory, however, open-space anxiety-like behavior in mice is tested in an elevated plus maze. The opaque walls and ceiling, size of the light chamber, and our specific light/dark transition test protocol is more specialized for detecting bright-space anxiety compared to the original version.Although the light/dark transition test and elevated plus maze are both used for assessing anxiety-like behavior, the results are not always consistent. For example, forebrain-specific calcineurin knockout mice spend an decreased amount of time in the light chamber in the light/dark transition test, but a increased amount of time in the open arms in the elevated plus maze(Miyakawa et al., 2003). We hypothesized that our light/dark transition test and elevated plus maze tests assess different aspects of anxiety-like behavior, such as bright-space anxiety and open-space anxiety-like behavior. To test this hypothesis, we used a large data set collected using the same mice in both tests. In our laboratory, we conduct behavioral test batteries covering many domains to screen for the behavioral significance of genes. The test battery requires several weeks to complete. During the testing period, mouse cages are kept on a rack in our facility. If mice are transported to our laboratory from other institutions, they are habituated to the environment of our facility for at least 1 week. After the habituation period, mice are subjected to a general health check and neurologic screening (Miyakawa et al., 2003), and after these simple examinations, mice are subjected to the light/dark transition test. The test is conducted in a sound proof experimental room after 30 min of habituation to the experimental room, as shown in the movie.We have assessed more than 60 strains of genetically engineered mutant mice using the protocol shown in the movie and have a large set of raw data for more than 3000 mice (including wild-type and mutant mice). We conducted factor analyses to take advantage of the large set of raw data. Indeed, we detected different factors from each test (unpublished data), although there were some significant correlations between indices (unpublished data).. Those data will be disclosed as part of a public database that we are now constructing.In our test battery, wild-type littermates are usually used as a control. As a background strain, C57BL/6J mice are commonly used. We collected the data of C57BL/6J mice used as a control of mutant mice in the test battery. The indices obtained from C57BL/6J mice in our light/dark transition test were as follows (n=795; mean SEM); distance traveled: 846.5 8.88 cm in the light chamber, 1478.5 9.53 cm in the dark chamber ; duration of time spent:215.8 1.85 s in the light, 395.8 1.86 s in the dark; number of transitions between compartments: 30.52 0.43; latency to enter the light chamber: 64.94 2.40 s. Movement of mice around the entrances to the chambers is detected by the camera and is counted as time spent in both chambers. Because of this, the sum of the mean duration of time spent in the light and that in dark compartments was greater than that of the entire duration of the test (600 s).In our protocol, a mouse is first placed in the dark chamber. Three seconds after placing the mouse in the dark chamber, the door between the chambers automatically opens and the mouse can move freely between the two chambers. C57BL/6J mice spend less time in the light chamber than in the dark chamber (p&lt;0.0001, duration if time spent in light: 215.8 1.85 s, duration of time spent in dark: 316.3 2.11 s [the time spent in the dark does not include the initial time spent in the dark prior to entering the light chamber for the first time], n=795, paired-t test). This indicates that C57BL/6J mice tend to avoid the light chamber, and that the time spent in the light is a good index of anxiety-like behavior.The results obtained from various strains of mutant mice are easily compared, because all data are obtained using the same protocol. To obtain phenotyping results of a strain of mutant mice that can be readily reproduced and compared with data of other mutants, the behavioral test methods should be as identical as possible between laboratories. Crabbe and colleagues tested the interaction between mouse behavior and laboratory environment using exactly the same inbred strains and one null mutant strain. In their study, the apparatus, test protocols, and many environmental variables were rigorously standardized. Despite the standardization, they found systematic differences in behavior across laboratories, although they also detected strain differences in all behaviors(Crabbe et al., 1999). They reported that uncontrollable variables and experiments characterizing mutants may yield results that are idiosyncratic to a particular laboratory. The procedural differences that exist between laboratories make it difficult to replicate or compare the results among laboratories. Here, we present our protocol for the light/dark transition test as a movie so that the details of the protocol can be demonstrated.Establishing visual documentation of the protocol will facilitate better understanding of the details of experimental procedures, allowing for standardization of the protocols used across laboratories and for comparisons of the behavioral phenotypes of various strains of mutant mice assessed using this test. Movies of other protocols, such as for the elevated plus maze, open field test, porsolt forced swim test, fear conditioning, that we use in our behavioral test battery are currently being made for publication as future video journal articles.

light/dark transition test for mice

Although all of the mouse genome sequences have been determined, we do not yet know the functions of most of these genes. Gene-targeting techniques, however, can be used to delete or manipulate a specific gene in mice. The influence of a given gene on a specific behavior can then be determined by conducting behavioral analyses of the mutant mice. As a test for behavioral phenotyping of mutant mice, the light/dark transition test is one of the most widely used tests to measure anxiety-like behavior in mice. The test is based on the natural aversion of mice to brightly illuminated areas and on their spontaneous exploratory behavior in novel environments. The test is sensitive to anxiolytic drug treatment. The apparatus consists of a dark chamber and a brightly illuminated chamber. Mice are allowed to move freely between the two chambers. The number of entries into the bright chamber and the duration of time spent there are indices of bright-space anxiety in mice. To obtain phenotyping results of a strain of mutant mice that can be readily reproduced and compared with those of other mutants, the behavioral test methods should be as identical as possible between laboratories. The procedural differences that exist between laboratories, however, make it difficult to replicate or compare the results among laboratories. Here, we present our protocol for the light/dark transition test as a movie so that the details of the protocol can be demonstrated. In our laboratory, we have assessed more than 60 strains of mutant mice using the protocol shown in the movie. Those data will be disclosed as a part of a public database that we are now constructing.

Visualization of the protocol will facilitate understanding of the details of the entire experimental procedure, allowing for standardization of the protocols used across laboratories and comparisons of the behavioral phenotypes of various strains of mutant mice assessed using this test.

Watch this Scientific Journal Video about Light/dark Transition Test for Mice at JoVE.com

Light/dark Transition Test for Mice

The light/dark transition test is one of the most widely used tests to measure anxiety-like behavior in mice. Here, we present a movie that shows detailed procedures on how we conduct the test.

Although all of the mouse genome sequences have been determined, we do not yet know the functions of most of these genes. Gene-targeting techniques, ...

Research

JoVE Journal

Biology

51.9K Aufrufe.  Graduate School of Medicine, Kyoto University. Der Hell / Dunkel-Übergangs-Test ist eine der am häufigsten verwendeten Tests zur Angst-ähnliches Verhalten bei Mäusen zu messen. Hier präsentieren wir einen Film, der detaillierte Verfahren, wie wir Verhalten der Test zeigt.

Serie: Light/dark Transition Test for Mice

Elevated Plus Maze for Mice

Although the mouse genome is now completely sequenced, the functions of most of the genes expressed in the brain are not known. The influence of a given gene on a specific behavior can be determined by behavioral analysis of mutant mice. If a target gene is expressed in the brain, behavioral phenotype of the mutant mice could elucidate the genetic mechanism of normal behaviors. The elevated plus maze test is one of the most widely used tests for measuring anxiety-like behavior. The test is based on the natural aversion of mice for open and elevated areas, as well as on their natural spontaneous exploratory behavior in novel environments. The apparatus consists of open arms and closed arms, crossed in the middle perpendicularly to each other, and a center area. Mice are given access to all of the arms and are allowed to move freely between them. The number of entries into the open arms and the time spent in the open arms are used as indices of open space-induced anxiety in mice. Unfortunately, the procedural differences that exist between laboratories make it difficult to duplicate and compare results among laboratories. Here, we present a detailed movie demonstrating our protocol for the elevated plus maze test. In our laboratory, we have assessed more than 90 strains of mutant mice using the protocol shown in the movie. These data will be disclosed as a part of a public database that we are now constructing.
Visualization of the protocol will promote better understanding of the details of the entire experimental procedure, allowing for standardization of the protocols used in different laboratories and comparisons of the behavioral phenotypes of various strains of mutant mice assessed using this test. 


The elevated plus maze test is one of the most widely used tests for measuring anxiety-like behavior in mice. Here, we present a movie showing the detailed procedures for conducting the test.

Although the mouse genome is now completely sequenced, the functions of most of the genes expressed in the brain are not known. The influence of a given ...

Murine Model for Parkinson's Disease: from 6-OH Dopamine Lesion to Behavioral Test

Parkinson's disease (PD) affects at least 6.5 million people worldwide, irrespective of gender, social, ethnic, economic, or geographic boundaries. Key symptoms, such as tremor, rigidity and bradikinesia, develop when about 3/4 of dopaminergic cells are lost in the substantia nigra, and fail to provide for the smooth, coordinated regulation of striatal motor circuits. Depression and hallucinations are common, and dementia eventually occurs in 20% of patients. At this time, there is no treatment to delay or stop the progression of PD. Rather, the medications currently available aim more towards the alleviation of these symptoms. New surgical strategies may reversibly switch on the functionally damaged circuits through the electrical stimulation of deep brain structures, but although deep brain stimulation is a major advance, it is not suitable for all patients. It remains therefore necessary to test new cell therapy approaches in preclinical models.
Selective neurotoxic disruption of dopaminergic pathways can be reproduced by injection of 6-hydroxydopamine (6-OHDA) or MPTP (1-methyl-4-phenyl-1,2,3,6-tertahydropyridine) whereas depleting drugs and oxidative-damaging chemicals may also reproduce specific features of PD in rodents. Unlike MPTP, 6-OHDA lesions cause massive irreversible neuronal loss, and can be uni- or bilateral. The 6-OHDA lesion model is reliable, leads to robust motor deficits, and is the most widely used after 40 years of research in rats1. As interactions between grafted cells and host can now be studied more thoroughly in mice rather than in rats, the model has been transposed to mice2,3, where it has been recently characterized4.
In this video, we demonstrate how to lesion the left nigro-striatal pathway of anesthetized mice by slowly delivering 2.0 &mu;L of 6-OHDA through a stereotaxically inserted micro-syringe needle. The loss of dopaminergic input occurs within days, and the functional impairments can be monitored over post-operative weeks and months by rating animal rotations induced by dopaminergic agents5. Here, we show full-body contralateral rotations occurring 10 minutes after a single subcutaneous administration of apomorphine, measured one month after the lesion. Outcomes and drawbacks are discussed below.

Murine Model for Parkinson&#039;s Disease: from 6-OH Dopamine Lesion to Behavioral Test

Parkinson disease is caused by loss of dopaminergic innervation to the striatum, which can be experimentally induced by 6-OH-dopamine. We describe how to perform a stereotaxic lesion and to monitor apomorphine-induced rotational behavior in mice. This model is useful and reliable for testing new therapies for Parkinson disease.

Parkinson's disease (PD) affects at least 6.5 million people worldwide, irrespective of gender, social, ethnic, economic, or geographic boundaries. Key ...

An Allele-specific Gene Expression Assay to Test the Functional Basis of Genetic Associations

The number of significant genetic associations with common complex traits is constantly increasing. However, most of these associations have not been understood at molecular level. One of the mechanisms mediating the effect of DNA variants on phenotypes is gene expression, which has been shown to be particularly relevant for complex traits1.
This method tests in a cellular context the effect of specific DNA sequences on gene expression. The principle is to measure the relative abundance of transcripts arising from the two alleles of a gene, analysing cells which carry one copy of the DNA sequences associated with disease (the risk variants)2,3. Therefore, the cells used for this method should meet two fundamental genotypic requirements: they have to be heterozygous both for DNA risk variants and for DNA markers, typically coding polymorphisms, which can distinguish transcripts based on their chromosomal origin (Figure 1). DNA risk variants and DNA markers do not need to have the same allele frequency but the phase (haplotypic) relationship of the genetic markers needs to be understood. It is also important to choose cell types which express the gene of interest. This protocol refers specifically to the procedure adopted to extract nucleic acids from fibroblasts but the method is equally applicable to other cells types including primary cells.
DNA and RNA are extracted from the selected cell lines and cDNA is generated. DNA and cDNA are analysed with a primer extension assay, designed to target the coding DNA markers4. The primer extension assay is carried out using the MassARRAY (Sequenom)5 platform according to the manufacturer's specifications. Primer extension products are then analysed by matrix-assisted laser desorption/ionization time of-flight mass spectrometry (MALDI-TOF/MS). Because the selected markers are heterozygous they will generate two peaks on the MS profiles. The area of each peak is proportional to the transcript abundance and can be measured with a function of the MassARRAY Typer software to generate an allelic ratio (allele 1: allele 2) calculation. The allelic ratio obtained for cDNA is normalized using that measured from genomic DNA, where the allelic ratio is expected to be 1:1 to correct for technical artifacts. Markers with a normalised allelic ratio significantly different to 1 indicate that the amount of transcript generated from the two chromosomes in the same cell is different, suggesting that the DNA variants associated with the phenotype have an effect on gene expression. Experimental controls should be used to confirm the results.

Genetic associations often remain unexplained at a functional level. This method aims to assess the effect of phenotype-associated genetic markers on gene expression by analyzing cells heterozygous for transcribed SNPs. The technology allows accurate measurement by MALDI-TOF mass spectrometry to quantify allele-specific primer extension products.

The number of significant genetic associations with common complex traits is constantly increasing. However, most of these associations have not been ...

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish water and sediment. The choice of biological indicators must be based on reliable scientific knowledge and, possibly, on the availability of standardized procedures. In this article, we present a standardized protocol that used the marine crustacean Artemia to evaluate the toxicity of chemicals and/or of marine environmental matrices. Scientists propose that the brine shrimp (Artemia) is a suitable candidate for the development of a standard bioassay for worldwide utilization. A number of papers have been published on the toxic effects of various chemicals and toxicants on brine shrimp (Artemia). The major advantage of this crustacean for toxicity studies is the overall availability of the dry cysts; these can be immediately used in testing and difficult cultivation is not demanded1,2. Cyst-based toxicity assays are cheap, continuously available, simple and reliable and are thus an important answer to routine needs of toxicity screening, for industrial monitoring requirements or for regulatory purposes3. The proposed method involves the mortality as an endpoint. The numbers of survivors were counted and percentage of deaths were calculated. Larvae were considered dead if they did not exhibit any internal or external movement during several seconds of observation4. This procedure was standardized testing a reference substance (Sodium Dodecyl Sulfate); some results are reported in this work. This article accompanies a video that describes the performance of procedural toxicity testing, showing all the steps related to the protocol.

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

This study concerns the development and standardization of a valuable methodological protocol to determine long-term (14 days) lethal toxicity exerted by chemical substances, industrial wastewater or sewage and liquid environmental samples on the saltwater crustacean, Artemia franciscana.

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish ...

Multi-parameter Measurement of the Permeability Transition Pore Opening in Isolated Mouse Heart Mitochondria

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with a molecular mass smaller than 1.5 kDa. Although the definitive molecular identity of the pore is still under debate, proteins such as cyclophilin D, VDAC and ANT contribute to mtPTP formation. While the involvement of mtPTP opening in cell death is well established1, accumulating evidence indicates that the mtPTP serves a physiologic role during mitochondrial Ca2+ homeostasis2, bioenergetics and redox signaling 3.
mtPTP opening is triggered by matrix Ca2+ but its activity can be modulated by several other factors such as oxidative stress, adenine nucleotide depletion, high concentrations of Pi, mitochondrial membrane depolarization or uncoupling, and long chain fatty acids4. In vitro, mtPTP opening can be achieved by increasing Ca2+ concentration inside the mitochondrial matrix through exogenous additions of Ca2+ (calcium retention capacity). When Ca2+ levels inside mitochondria reach a certain threshold, the mtPTP opens and facilitates Ca2+ release, dissipation of the proton motive force, membrane potential collapse and an increase in mitochondrial matrix volume (swelling) that ultimately leads to the rupture of the outer mitochondrial membrane and irreversible loss of organelle function.
Here we describe a fluorometric assay that allows for a comprehensive characterization of mtPTP opening in isolated mouse heart mitochondria. The assay involves the simultaneous measurement of 3 mitochondrial parameters that are altered when mtPTP opening occurs: mitochondrial Ca2+ handling (uptake and release, as measured by Ca2+ concentration in the assay medium), mitochondrial membrane potential, and mitochondrial volume. The dyes employed for Ca2+ measurement in the assay medium and mitochondrial membrane potential are Fura FF, a membrane impermeant, ratiometric indicator which undergoes a shift in the excitation wavelength in the presence of Ca2+, and JC-1, a cationic, ratiometric indicator which forms green monomers or red aggregates at low and high membrane potential, respectively. Changes in mitochondrial volume are measured by recording light scattering by the mitochondrial suspension. Since high-quality, functional mitochondria are required for the mtPTP opening assay, we also describe the steps necessary to obtain intact, highly coupled and functional isolated heart mitochondria.

A spectrofluorometric protocol for the measurement of the mitochondrial permeability transition pore opening in isolated mouse heart mitochondria is presented here. The assay involves the simultaneous measurement of mitochondria Ca2+ handling, mitochondrial membrane potential and mitochondrial volume. The procedure for obtaining high-quality and functional heart mitochondria is also described.

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with ...

Blastomere Explants to Test for Cell Fate Commitment During Embryonic Development

Fate maps, constructed from lineage tracing all of the cells of an embryo, reveal which tissues descend from each cell of the embryo. Although fate maps are very useful for identifying the precursors of an organ and for elucidating the developmental path by which the descendant cells populate that organ in the normal embryo, they do not illustrate the full developmental potential of a precursor cell or identify the mechanisms by which its fate is determined. To test for cell fate commitment, one compares a cell's normal repertoire of descendants in the intact embryo (the fate map) with those expressed after an experimental manipulation. Is the cell's fate fixed (committed) regardless of the surrounding cellular environment, or is it influenced by external factors provided by its neighbors? Using the comprehensive fate maps of the Xenopus embryo, we describe how to identify, isolate and culture single cleavage stage precursors, called blastomeres. This approach allows one to assess whether these early cells are committed to the fate they acquire in their normal environment in the intact embryo, require interactions with their neighboring cells, or can be influenced to express alternate fates if exposed to other types of signals.

The fate of an individual embryonic cell can be influenced by inherited molecules and/or by signals from neighboring cells. Utilizing fate maps of the cleavage stage Xenopus embryo, single blastomeres can be identified for culture in isolation to assess the contributions of inherited molecules versus cell-cell interactions.

Fate maps, constructed from lineage tracing all of the cells of an embryo, reveal  which tissues descend from each cell of the embryo.  Although fate maps ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Test Samples for Optimizing STORM Super-Resolution Microscopy

STORM is a recently developed super-resolution microscopy technique with up to 10 times better resolution than standard fluorescence microscopy techniques. However, as the image is acquired in a very different way than normal, by building up an image molecule-by-molecule, there are some significant challenges for users in trying to optimize their image acquisition. In order to aid this process and gain more insight into how STORM works we present the preparation of 3 test samples and the methodology of acquiring and processing STORM super-resolution images with typical resolutions of between 30-50 nm. By combining the test samples with the use of the freely available rainSTORM processing software it is possible to obtain a great deal of information about image quality and resolution. Using these metrics it is then possible to optimize the imaging procedure from the optics, to sample preparation, dye choice, buffer conditions, and image acquisition settings. We also show examples of some common problems that result in poor image quality, such as lateral drift, where the sample moves during image acquisition and density related problems resulting in the 'mislocalization' phenomenon.

We describe the preparation of three test samples and how they can be used to optimize and assess the performance of STORM microscopes. Using these examples we show how to acquire raw data and then process it to acquire super-resolution images in cells of approximately 30-50 nm resolution.

STORM is a recently developed super-resolution microscopy technique with up to 10 times better resolution than standard fluorescence microscopy ...

Detection of Alternative Splicing During Epithelial-Mesenchymal Transition

Alternative splicing plays a critical role in the epithelial-mesenchymal transition (EMT), an essential cellular program that occurs in various physiological and pathological processes. Here we describe a strategy to detect alternative splicing during EMT using an inducible EMT model by expressing the transcription repressor Twist. EMT is monitored by changes in cell morphology, loss of E-cadherin localization at cell-cell junctions, and the switched expression of EMT markers, such as loss of epithelial markers E-cadherin and &#947;-catenin and gain of mesenchymal markers N-cadherin and vimentin. Using isoform-specific primer sets, the alternative splicing of interested mRNAs are analyzed by quantitative RT-PCR. The production of corresponding protein isoforms is validated by immunoblotting assays. The method of detecting splice isoforms described here is also suitable for the study of alternative splicing in other biological processes.

Alternative splicing regulation has been shown to contribute to the epithelial-mesenchymal transition (EMT), an essential cellular program in various physiological and pathological processes. Here we describe a method utilizing an inducible EMT model for the detection of alternative splicing during EMT.

Alternative splicing plays a critical role in the epithelial-mesenchymal transition (EMT), an essential cellular program that occurs in various ...

Intravenous Injections in Neonatal Mice

Intravenous injection is a clinically applicable manner to deliver therapeutics. For adult rodents and larger animals, intravenous injections are technically feasible and routine. However, some mouse models can have early onset of disease with a rapid progression that makes administration of potential therapies difficult. The temporal (or facial) vein is just anterior to the ear bud in mice and is clearly visible for the first two days after birth on either side of the head using a dissecting microscope. During this window, the temporal vein can be injected with volumes up to 50 &#956;l. The injection is safe and well tolerated by both the pups and the dams. A typical injection procedure is completed within 1-2 min, after which the pup is returned to the home cage. By the third postnatal day the vein is difficult to visualize and the injection procedure becomes technically unreliable. This technique has been used for delivery of adeno-associated virus (AAV) vectors, which in turn can provide almost body-wide, stable transgene expression for the life of the animal depending on the viral serotype chosen.

Animal models of pediatric disease can experience early onset and aggressive disease progression. Clinically relevant therapy delivery to young mouse models can be technically difficult. This protocol describes a non-invasive intravenous injection method for newborn mice within the first two postnatal days of life.