We would like to thank the members of the Skok lab, especially Susannah Hewitt, for discussions and comments. This work is supported by the National Institute of Health grants R01GM086852, RC1CA145746 (J.A.S.). J.A.S. is a Leukemia &amp; Lymphoma Society scholar. J.C. is an Irvington Institute Fellow of the Cancer Research Institute. M.M. is supported by a National Science Foundation Grant Integrative Graduate Education and Research Traineeship (NSF IGERT 0333389).

1. DNA Probe Labeling with Fluorophores: Nick Translation (~ 6 hr)
<ol>
 <li>Clean BAC DNA (prepared by maxi-prep) or plasmids or PCR products, all resuspended in H2O, can be used for labeling. Note that for a robust FISH signal, probes should span at least 10 kb.</li>
 <li>Incubate DNA in RNase A for 30 min at 37 &deg;C (All reagents are listed in Table 1).</li>
 <li>Incubate the nick translation reaction for 2 hr at 16 &deg;C (see Tables 2 and 3). Alternative methods of direct labeling may be used (Example: FISH Tag, Invitrogen).</li>
 <li>Inactivate the reaction for one hr at -80 &deg;C or overnight at -20 &deg;C.</li>
 <li>Determine probe size on a 2% agarose gel. The smear should be between 100 and 1,000 bp, with the majority at approximately 300-500 bp (Note that the smear of Cy5-probes is not visible). If the DNA is not digested enough, more DNA Pol I and DNase I can be added to the reaction and incubated for two more hr at 16 &deg;C.</li>
 <li>Purify probes on 0.025 mm filters in two liter beakers filled with H20 for two hr in the dark.</li>
 <li>Add blocking factors to the probes as follows: 10 &mu;g of Cot-1 DNA, 10 &mu;g of Hybloc DNA and 100 &mu;g of salmon sperm for 10 &mu;g of DNA probes. Store DNA probes at -20 &deg;C.</li>
</ol>
2. Probe Precipitation/Denaturation/Pre-annealing (3-4 hr)
<ol>
 <li>Between 0.3 &mu;g and 1 &mu;g of DNA probe is used per coverslip. Mix probes with 0.1 volume of 3M NaAc (pH 5.2) and 2.5 volumes of 100% ethanol, incubate for one hr at -80 &deg;C or overnight at -20 &deg;C and spin down at 13,000 rpm at 4 &deg;C for 30 min. Note that the amount of DNA probe may be adjusted depending on the quality of the DNA and the nuclear region of hybridization. Alternative methods of direct labeling may allow the use of reduced amounts of probe. For detection of multiple DNA FISH signals on one slide, the different probes can be precipitated together (except probes that should not be pre-annealed, like repeat sequences). Probes for several experiments performed at the same time can also be precipitated together. Pellets should be lightly colored according to the fluorophores used (if small amounts of probes are precipitated, the pellet may not be visible).</li>
 <li>Dry pellets and resuspend them in 15 &mu;l hybridization buffer (see Table 4) per coverslip with shaking (using an Eppendorf thermomixer) in the dark at 37-45 &deg;C (for approximately 20 min).</li>
 <li>Denature probes for 5 min at 75-95 &deg;C.</li>
 <li>Pre-anneal probes at 37 &deg;C for 30-60 min. Length of pre-annealing may be adjusted depending on the complexity and quality of the probes.</li>
 <li>Apply probes to the coverslips for overnight hybridization (see Section 3).</li>
</ol>
3. 3-dimensional DNA FISH - First Day (~ 3 hr)
<ol>
 <li>Non-adherent cells are concentrated in a small volume of 1x PBS (~ 2x105 cells in 5-10 &mu;l 1x PBS per coverslip) and dropped on the center of a poly-L-lysine coated coverslip. Adherent cells should be cultured on coverslips for at least 24 hr (~ 70% confluence) (coverslips may be coated with 0.1% gelatin). Square 18x18 to 24-24 mm coverslips are placed in 6-well plates, and throughout the protocol, two ml of each solution is used per well (alternatively round 1.5 mm coverslips can be placed in 12/24-well plates).</li>
 <li>Fix cells in 2% paraformaldehyde / 1x PBS, pH 7-7.4, for 10 min at RT (4% PFA stocks can be aliquoted, stored at -20 &deg;C). Paraformaldehyde can be purchased in solution (16%), or 4% stock solutions can be prepared from powder/prills. Paraformaldehyde powder is dissolved in 1x PBS at &gt; 60 &deg;C (pH may be increased to 8-9 to facilitate dissolution), cooled-down on ice, adjusted to pH 7-7.4, filtered, aliquoted in falcons and stored for weeks at -20 &deg;C. </li>
 <li>After three rinses in 1x PBS, permeabilize cells in iced-cold 0.4% Triton-X-100 / 1x PBS for 5 min on ice. Length of permeabilization may be adjusted depending on the cell-type. N.B.: If DNA FISH is combined with immunofluorescence, the immunostaining should be performed at this stage (see Section 4).</li>
 <li>After three rinses in 1x PBS, incubate cells with 0.1 &mu;g/&mu;l RNase A in 1x PBS for one hr at 37 &deg;C. Coverslips are placed cell-side down onto a drop of 100 &mu;l of solution on parafilm or a slide in a humid chamber. Coverslips are then carefully removed with forceps. If any resistance is encountered, coverslips should be flooded with 1x PBS in order to avoid damaging the cells.</li>
 <li>After three rinses in 1x PBS, permeabilize cells in iced-cold 0.7% Triton-X-100 / 0.1 M HCl for 10 min on ice.</li>
 <li>After three rinses in 1x PBS, denature cells in 1.9 M HCl for 30 min at RT. Alternately, cells can be denatured in 50% formamide / 2x SSC at 75-80 &deg;C for at least 30 min. Note that hybridization length (and temperature) may be optimized depending on the cell-type.</li>
 <li>After three rinses in ice-cold 1x PBS, hybridize cells with probes overnight at 37 &deg;C in a dark and humid chamber. Coverslips are placed cell-side down onto a drop of 15 &mu;l of probe on a slide, and sealed with rubber cement.</li>
</ol>
4. 3-dimensional Immuno-FISH - First Day (~ 6-7 hr)
<ol>
 <li>For combined immunofluorescence /DNA FISH, immuno-staining should be performed after the permeabilization in 0.4% Triton-X-100 (Step 3.3).</li>
 <li>Incubate cells in blocking solution for 30 min at RT (2.5% bovine serum albumin (BSA), 10% Normal Goat Serum, 0.1% Tween-20). The blocking solution is filtered, aliquoted in 1.5 ml Eppendorfs and stored for months at -20 &deg;C.</li>
 <li>Incubate cells with the primary antibody raised against the protein or the histone modification of interest diluted in blocking solution, for one hr at RT in a humid chamber. Here we show the example of an antibody against phosphorylated serine-139 of H2AX (&gamma;-H2AX, Millipore; See Table 5 for details). Coverslips are placed cell-side down onto a drop of 100 &mu;l of solution on parafilm or a slide in a humid chamber. Coverslips are then carefully removed with forceps (flooded with 1x PBS if necessary).Note that dilution and incubation length may be adjusted depending on the quality of the antibody and cell-types.</li>
 <li>Wash cells in 0.2% BSA / 0.1% Tween-20 / 1x PBS, three times 5 min at RT with shaking.</li>
 <li>Incubate cells with the appropriate fluorophore-conjugated secondary antibody diluted in blocking solution for one hr at RT in a dark and humid chamber. Here we show an example of detection with a secondary goat anti-mouse antibody (Alexa Fluor 488 or 555, Invitrogen; See Table 5 for details). Hapten-conjugated secondary antibodies (biotin, digoxigenin, etc) could also be used. In this case, an extra step for appropriate detection (streptavidin, anti-dig, etc) can be performed either before or after over-night DNA-FISH hybridization.</li>
 <li>Rinse cells in 0.1% Tween-20 / 1x PBS, three times 5 min at RT with shaking in the dark.</li>
 <li>Post-fix cells in 2% paraformaldehyde / 1x PBS for 10 min at RT in the dark.</li>
 <li>Continue DNA FISH as above from incubation in RNase A (Step 3.4).</li>
</ol>
5. 3-dimensional DNA FISH / Immuno-FISH - Second Day (~ 2 hr)
<ol>
 <li>Carefully remove rubber cement, flood coverslips with 2x SSC, carefully remove with forceps and place them in wells containing washing solution.</li>
 <li>Wash cells in 2x SSC for 30 min at 37 &deg;C in the dark with shaking.</li>
 <li>Wash cells in 2x SSC for 30 min at RT in the dark with shaking.</li>
 <li>Wash cells in 1x SSC for 30 min at RT in the dark with shaking. Note that in case of denaturation with formamide, washes should be replaced by the following: 3 washes in 50% formamide / 2x SSC and 3 washes in 2x SSC, 5 min each, at 37 &deg;C.</li>
 <li>Mount cells in ProLong Gold mounting medium containing 1.5 &mu;g/ml DAPI (4,6-diamidino-2-phenylindole) for counterstaining of total DNA. Coverslips are placed cell-side down onto a drop of 10-15 &mu;l of mounting medium on a slide and sealed with nail polish. Slides are kept for months at -20 &deg;C.</li>
</ol>
6. 3D Microscopy and Analysis
<ol>
 <li>3D images can be acquired with different microscope systems. In our experiments, optical sections separated by 0.3 &mu;m are collected on a Leica SP5 AOBS confocal system (Acousto-Optical Beam Splitter). Note that the separation between optical planes may be adjusted depending on the type of analysis.</li>
 <li>3D image stacks can be analyzed using different software and tools. We use the Image J software to assess distance measurements between loci of interest and to analyze different types of association of the loci of interest with sub-nuclear compartments or proteins.</li>
 <li>All statistical analyses are performed to compare two (or more) different biological conditions with pairwise assessment(s) of significance: comparison between different cell types or stages, comparison between different loci of interest. In all statistical tests, P-values &le; 5.00E-2 (a = 0.05) are taken to be significant (1.00E-2 &le; P &le; 5.00E-2 * significant; 1.00E-3 &le; P &le; 1.00E-2 ** very significant; P &lt; 1.00E-3 *** highly significant).</li>
</ol>
Statistical analysis of the entire distributions of distances between two alleles. We first construct cumulative distribution frequency curves over the entire range of measured distances between the two alleles.To assess the significance of differences in the distributions of empirical inter-allelic distances we use the nonparametric two-sample Kolmogorov-Smirnov (KS) test 13,14 .
Statistical analysis of close association (i.e. pairing) of two alleles using an optimal distance cut-off. To identify the range of most robust differences in distances between two alleles or loci, we use a series of two-tail Fisher exact test 15 s. Namely, at each measured distance we test whether one condition is significantly over-represented at shorter distances between the two samples. The minimum in the distribution of P-Values indicates the range of distances that should be considered as a cut-off for close association (i.e. pairing) of the two alleles. Subsequently the two-tail Fisher exact test is used to assess the significance of pairing of two alleles at the identified cut-off value15. Multiple testing corrections are applied to account for the total number of Fisher tests performed 16.
Statistical analysis of association of the locus of interest with sub-nuclear compartments or proteins. The two-tail Fisher-exact test is used to analyze the significance of association of the locus of interest with different compartments or proteins 15.

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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differences+in+the+localization+and+morphology+of+chromosomes+in+the+human+nucleus.">Differences in the localization and morphology of chromosomes in the human nucleus.</a> The Journal of cell biology. 145, 1119-1131 (1999).</li><li>Chaumeil, J., Le Baccon, P., Wutz, A., Heard, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+role+for+Xist+RNA+in+the+formation+of+a+repressive+nuclear+compartment+into+which+genes+are+recruited+when+silenced.">A novel role for Xist RNA in the formation of a repressive nuclear compartment into which genes are recruited when silenced.</a> Genes Dev. 20, 2223-2237 (2006).</li><li>Walter, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Towards+many+colors+in+FISH+on+3D-preserved+interphase+nuclei.">Towards many colors in FISH on 3D-preserved interphase nuclei.</a> Cytogenetic and genome research. 114, 367-378 (2006).</li><li>Toomre, D., Bewersdorf, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+wave+of+cellular+imaging.">A new wave of cellular imaging.</a> Annual review of cell and developmental biology. 26, 285-314 (2010).</li><li>Schermelleh, L., Heintzmann, R., Leonhardt, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+guide+to+super-resolution+fluorescence+microscopy.">A guide to super-resolution fluorescence microscopy.</a> The Journal of cell biology. 190, 165-175 (2010).</li><li>Dobbie, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=OMX:+a+new+platform+for+multimodal,+multichannel+wide-field+imaging.">OMX: a new platform for multimodal, multichannel wide-field imaging.</a> Cold Spring Harbor protocols. , 899-909 (2011).</li><li>Boyle, S., Rodesch, M. J., Halvensleben, H. A., Jeddeloh, J. A., Bickmore, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+in+situ+hybridization+with+high-complexity+repeat-free+oligonucleotide+probes+generated+by+massively+parallel+synthesis.">Fluorescence in situ hybridization with high-complexity repeat-free oligonucleotide probes generated by massively parallel synthesis.</a> Chromosome research : an international journal on the molecular, supramolecular and evolutionary aspects of chromosome biology. 19, 901-909 (2011).</li></ol>

The authors declare that they have no competing financial interests.

The techniques detailed above were used in our lab to analyze the regulation of V(D)J recombination of the Immunoglobulin and Tcra/d loci in developing lymphocytes 30,31 . We are confident that this technique can be adapted for detection of various nuclear proteins, nuclear compartments and loci, in different cell-types. Modifications of the protocol may be necessary, and in this case the major steps to focus on are the following. First, the length of permeabilization can be adjusted de...

Epigenetic mechanisms trigger establishment and inheritance of developmental and cell-type specific transcriptional profiles. At one level this involves modulation of chromatin packaging that defines active or silent genomic regions. On a larger scale, global 3D organization of the genome and nuclear architecture also play a role in the control of transcriptional patterns. Thus, dissection of these epigenomic features is essential for a full understanding of how genes are regulated 6-11.
Combined immunofluorescence and 3D DNA FISH provide a unique opportunity to complement molecular and biochemical analyses by assessing specific interactions/associations of DNA sequences and/or proteins within the nucleus. Furthermore, while genome-wide high throughput techniques such as chromatin immunoprecipitation (ChIP-seq) or chromosome capture conformation coupled with deep sequencing (4C-seq, 5C, Hi-C) provide global data on cell populations12, immunofluorescence/DNA FISH techniques enable analyses at the single cell level.
Here we describe a protocol for simultaneous detection of histone modifications by immunofluorescence and DNA sequences by DNA FISH followed by 3D microscopy and analyses (3D immuno-FISH). The advantage of this protocol is the combined visualization of DNA and preservation of protein structures. Our experience in this field has enabled us to improve and simplify existing protocols. Although we have used this protocol to detect DNA double-stranded breaks in lymphocytes undergoing recombination, this method can be applied to other proteins and other cell-types.

<table border="1">
 <tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalogue Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>H2O</td>
 <td>Fisher</td>
 <td># BP2470</td>
 <td></td>
 </tr>
 <tr>
 <td>RNase A</td>
 <td>Sigma</td>
 <td># R4642</td>
 <td></td>
 </tr>
 <tr>
 <td>dNTP</td>
 <td>Sigma</td>
 <td># DNTP100</td>
 <td></td>
 </tr>
 <tr>
 <td>Alexa dUTP</td>
 <td>Invitrogen</td>
 <td># C11397 to C-11401</td>
 <td></td>
 </tr>
 <tr>
 <td>Cy3 or Cy5 dUTP</td>
 <td>Fisher</td>
 <td># 45-001-xxx</td>
 <td></td>
 </tr>
 <tr>
 <td>DNase I</td>
 <td>Roche</td>
 <td># 04536282001</td>
 <td></td>
 </tr>
 <tr>
 <td>DNA Pol I</td>
 <td>Biolabs</td>
 <td># M0209</td>
 <td></td>
 </tr>
 <tr>
 <td>0.025 &mu;m filters</td>
 <td>Millipore</td>
 <td># VSWP02500</td>
 <td></td>
 </tr>
 <tr>
 <td>Cot-1 DNA 1 mg/ml</td>
 <td>Invitrogen</td>
 <td># 18440</td>
 <td></td>
 </tr>
 <tr>
 <td>Hybloc DNA 1 mg/ml</td>
 <td>Applied Genetics</td>
 <td># MHB</td>
 <td></td>
 </tr>
 <tr>
 <td>Salmon sperm</td>
 <td>Sigma</td>
 <td># D1626</td>
 <td>powder to be resuspended at 10 mg/ml in H2O</td>
 </tr>
 <tr>
 <td>NaAc (Sodium Acetate, pH 5.2, buffer solution)</td>
 <td>Sigma</td>
 <td># S7899</td>
 <td></td>
 </tr>
 <tr>
 <td>Ficoll 400 (Mol Biol grade)</td>
 <td>Fisher</td>
 <td># 525</td>
 <td></td>
 </tr>
 <tr>
 <td>Polyvinylpyrrolidone (Mol Biol grade)</td>
 <td>Fisher</td>
 <td># BP431</td>
 <td></td>
 </tr>
 <tr>
 <td>Dextran sulfate powder</td>
 <td>Sigma</td>
 <td># D8906</td>
 <td></td>
 </tr>
 <tr>
 <td>SSPE (Saline-Sodium Phosphate-EDTA) 20x solution</td>
 <td>Fisher</td>
 <td># BP1328</td>
 <td></td>
 </tr>
 <tr>
 <td>Formamide</td>
 <td>Fisher</td>
 <td># BP227</td>
 <td></td>
 </tr>
 <tr>
 <td>Coverslips</td>
 <td>Fisher</td>
 <td># 12-548-B</td>
 <td></td>
 </tr>
 <tr>
 <td>Slides</td>
 <td>Fisher</td>
 <td># 12-550</td>
 <td></td>
 </tr>
 <tr>
 <td>6-well plates</td>
 <td>Fisher</td>
 <td># 0720080</td>
 <td></td>
 </tr>
 <tr>
 <td>PBS, 10x</td>
 <td>Fisher</td>
 <td># MT-46-013-CM</td>
 <td></td>
 </tr>
 <tr>
 <td>Poly-L-lysine solution</td>
 <td>Sigma</td>
 <td># P8920</td>
 <td></td>
 </tr>
 <tr>
 <td>Paraformaldehyde, prills, 95%</td>
 <td>Sigma</td>
 <td># 441244</td>
 <td></td>
 </tr>
 <tr>
 <td>Triton-X-100, Mol Biol grade</td>
 <td>Sigma</td>
 <td># T8787</td>
 <td></td>
 </tr>
 <tr>
 <td>BSA (Bovine Serum Albumin) Fraction V</td>
 <td>Fisher</td>
 <td># BP 1600</td>
 <td></td>
 </tr>
 <tr>
 <td>Normal goat serum</td>
 <td>Vector Labs</td>
 <td># S-1000</td>
 <td></td>
 </tr>
 <tr>
 <td>Tween-20, Mol Biol grade</td>
 <td>Sigma</td>
 <td># P9416</td>
 <td></td>
 </tr>
 <tr>
 <td>SSC (Saline Sodium Citrate) 20x solution</td>
 <td>Fisher</td>
 <td># BP1325</td>
 <td></td>
 </tr>
 <tr>
 <td>ProLong Gold antifade reagent</td>
 <td>Invitrogen</td>
 <td># P36930</td>
 <td></td>
 </tr>
 <tr>
 <td>DAPI (4',6-diamidino-2-phenylindole)</td>
 <td>Sigma</td>
 <td># D9542</td>
 <td></td>
 </tr>
 <tr>
 <td>Best test one coat rubber cement</td>
 <td>Art or office supply stores</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 	<td></td>
 <td></td>
 <td></td>
 <td>Table 1. Specific reagents and small equipment.</td>
 </tr>
 </tbody>
</table>

combined immunofluorescence and dna fish on 3d-preserved interphase nuclei to study changes in 3d nuclear organization

Fluorescent in situ hybridization using DNA probes on 3-dimensionally preserved nuclei followed by 3D confocal microscopy (3D DNA FISH) represents the most direct way to visualize the location of gene loci, chromosomal sub-regions or entire territories in individual cells. This type of analysis provides insight into the global architecture of the nucleus as well as the behavior of specific genomic loci and regions within the nuclear space. Immunofluorescence, on the other hand, permits the detection of nuclear proteins (modified histones, histone variants and modifiers, transcription machinery and factors, nuclear sub-compartments, etc). The major challenge in combining immunofluorescence and 3D DNA FISH is, on the one hand to preserve the epitope detected by the antibody as well as the 3D architecture of the nucleus, and on the other hand, to allow the penetration of the DNA probe to detect gene loci or chromosome territories 1-5. Here we provide a protocol that combines visualization of chromatin modifications with genomic loci in 3D preserved nuclei.

DNA and immuno-FISH are used in the Skok lab to study the changes in nuclear organization associated with the process of V(D)J recombination of antigen receptor loci during B and T lymphocyte development. The techniques detailed above enable us to i) measure distances between the two ends of a locus (contraction) ii) measure distances between alleles or loci (pairing), iii) analyze the DNA damage occurring within loci, iv) assess the location of alleles and loci relative to nuclear sub-compartments (repressive per...

Watch this Scientific Journal Video about Combined Immunofluorescence and DNA FISH on 3D-preserved Interphase Nuclei to Study Changes in 3D Nuclear Organization at JoVE.com

Combined Immunofluorescence and DNA FISH on 3D-preserved Interphase Nuclei to Study Changes in 3D Nuclear Organization

Here we describe a protocol for simultaneous detection of histone modifications by immunofluorescence and DNA sequences by DNA FISH followed by 3D microscopy and analyses (3D immuno-DNA FISH).

Fluorescent in situ hybridization using DNA probes  on 3-dimensionally preserved nuclei followed by 3D confocal microscopy (3D DNA  FISH) represents the ...

combined-immunofluorescence-dna-fish-on-3d-preserved-interphase

Research

JoVE Journal

Biology

18.2K visualizações.  New York University School of Medicine. Aqui nós descrevemos um protocolo para a detecção simultânea de modificações de histonas por imunofluorescência e seqüências de DNA por FISH DNA seguido por microscopia 3D e análises (3D FISH imuno-DNA).

Vídeo: Combined Immunofluorescence and DNA FISH on 3D-preserved Interphase Nuclei to Study Changes in 3D Nuclear Organization

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

DNA-based Fish Species Identification Protocol

We have developed a fast, simple, and accurate DNA-based screening method to identify the fish species present in fresh and processed seafood samples. This versatile method employs PCR amplification of genomic DNA extracted from fish samples, followed by restriction fragment length polymorphism (RFLP) analysis to generate fragment patterns that can be resolved on the Agilent 2100 Bioanalyzer and matched to the correct species using RFLP pattern matching software.
The fish identification method uses a simple, reliable, spin column- based protocol to isolate DNA from fish samples. The samples are treated with proteinase K to release the nucleic acids into solution. DNA is then isolated by suspending the sample in binding buffer and loading onto a micro- spin cup containing a silica- based fiber matrix. The nucleic acids in the sample bind to the fiber matrix. The immobilized nucleic acids are washed to remove contaminants, and total DNA is recovered in a final volume of 100 &mu;l. The isolated DNA is ready for PCR amplification with the provided primers that bind to sequences found in all fish genomes. The PCR products are then digested with three different restriction enzymes and resolved on the Agilent 2100 Bioanalyzer. The fragment lengths produced in the digestion reactions can be used to determine the species of fish from which the DNA sample was prepared, using the RFLP pattern matching software containing a database of experimentally- derived RFLP patterns from commercially relevant fish species.

This publication describes how to use the Agilent Fish Species Identification System to identify the species of a fish by extracting DNA and performing PCR and RFLP analysis.

We have developed a fast, simple, and accurate DNA-based screening method to identify the fish species present in fresh and processed seafood samples. ...

Robust 3D DNA FISH Using Directly Labeled Probes

3D DNA FISH has become a major tool for analyzing three-dimensional organization of the nucleus, and several variations of the technique have been published. In this article we describe a protocol which has been optimized for robustness, reproducibility, and ease of use. Brightly fluorescent directly labeled probes are generated by nick-translation with amino-allyldUTP followed by chemical coupling of the dye. 3D DNA FISH is performed using a freeze-thaw step for cell permeabilization and a heating step for simultaneous denaturation of probe and nuclear DNA. The protocol is applicable to a range of cell types and a variety of probes (BACs, plasmids, fosmids, or Whole Chromosome Paints) and allows for high-throughput automated imaging. With this method we routinely investigate nuclear localization of up to three chromosomal regions.

We describe a robust and versatile protocol for analyzing nuclear architecture by 3D DNA FISH using directly labeled fluorescent probes.

3D DNA FISH has become a major tool for  analyzing three-dimensional organization of the nucleus, and several variations  of the technique have been ...

Assessing Species-specific Contributions To Craniofacial Development Using Quail-duck Chimeras

The generation of chimeric embryos is a widespread and powerful approach to study cell fates, tissue interactions, and species-specific contributions to the histological and morphological development of vertebrate embryos. In particular, the use of chimeric embryos has established the importance of neural crest in directing the species-specific morphology of the craniofacial complex. The method described herein utilizes two avian species, duck and quail, with remarkably different craniofacial morphology. This method greatly facilitates the investigation of molecular and cellular regulation of species-specific pattern in the craniofacial complex.&#160;Experiments in quail and duck chimeric embryos have already revealed neural crest-mediated tissue interactions and cell-autonomous behaviors that regulate species-specific pattern in the craniofacial skeleton, musculature, and integument. The great diversity of neural crest derivatives suggests significant potential for future applications of the quail-duck chimeric system to understanding vertebrate development, disease, and evolution.

This article describes a method to generate chimeric embryos that are&#160;designed to test the species-specific contributions of neural crest and/or other tissues to craniofacial development.

The generation of chimeric embryos is a widespread and powerful approach to study cell fates, tissue interactions, and species-specific contributions to ...

Combined DNA-RNA Fluorescent In situ Hybridization (FISH) to Study X Chromosome Inactivation in Differentiated Female Mouse Embryonic Stem Cells

Fluorescent in situ hybridization (FISH) is a molecular technique which enables the detection of nucleic acids in cells. DNA FISH is often used in cytogenetics and cancer diagnostics, and can detect aberrations of the genome, which often has important clinical implications. RNA FISH can be used to detect RNA molecules in cells and has provided important insights in regulation of gene expression. Combining DNA and RNA FISH within the same cell is technically challenging, as conditions suitable for DNA FISH might be too harsh for fragile, single stranded RNA molecules. We here present an easily applicable protocol which enables the combined, simultaneous detection of Xist RNA and DNA encoded by the X chromosomes. This combined DNA-RNA FISH protocol can likely be applied to other systems where both RNA and DNA need to be detected.

Combined DNA-RNA Fluorescent In situ Hybridization FISH to Study X Chromosome Inactivation in Differentiated Female Mouse Embryonic Stem Cells

Fluorescent in situ hybridization (FISH) allows the detection of nucleic acids in their native environment within cells. We here describe a protocol for the combined, simultaneous detection of RNA and DNA by means of FISH, which can be used to study X chromosome inactivation in mouse embryonic stem cells.

Fluorescent in situ hybridization (FISH) is a molecular technique which enables the detection of nucleic acids in cells. DNA FISH is often used in ...

Measuring DNA Damage and Repair in Mouse Splenocytes After Chronic In Vivo Exposure to Very Low Doses of Beta- and Gamma-Radiation

Low dose radiation exposure may produce a variety of biological effects that are different in quantity and quality from the effects produced by high radiation doses. Addressing questions related to environmental, occupational and public health safety in a proper and scientifically justified manner heavily relies on the ability to accurately measure the biological effects of low dose pollutants, such as ionizing radiation and chemical substances. DNA damage and repair are the most important early indicators of health risks due to their potential long term consequences, such as cancer. Here we describe a protocol to study the effect of chronic in vivo exposure to low doses of &#947;- and &#946;-radiation on DNA damage and repair in mouse spleen cells. Using a commonly accepted marker of DNA double-strand breaks, phosphorylated histone H2AX called &#947;H2AX, we demonstrate how it can be used to evaluate not only the levels of DNA damage, but also changes in the DNA repair capacity potentially produced by low dose in vivo exposures. Flow cytometry allows fast, accurate and reliable measurement of immunofluorescently labeled &#947;H2AX in a large number of samples. DNA double-strand break repair can be evaluated by exposing extracted splenocytes to a challenging dose of 2 Gy to produce a sufficient number of DNA breaks to trigger repair and by measuring the induced (1 hr post-irradiation) and residual DNA damage (24 hrs post-irradiation). Residual DNA damage would be indicative of incomplete repair and the risk of long-term genomic instability and cancer. Combined with other assays and end-points that can easily be measured in such in vivo studies (e.g., chromosomal aberrations, micronuclei frequencies in bone marrow reticulocytes, gene expression, etc.), this approach allows an accurate and contextual evaluation of the biological effects of low level stressors.

Measuring DNA Damage and Repair in Mouse Splenocytes After Chronic In Vivo Exposure to Very Low Doses of Beta- and Gamma-Radiation

A protocol to evaluate changes in DNA damage levels and DNA repair capacity that may be induced by chronic in vivo low dose irradiation in mouse spleen lymphocytes, by measuring phosphorylated histone H2AX, a marker of DNA double-strand breaks, using flow cytometry is presented.

Low dose radiation exposure may produce a variety of biological effects that are different in quantity and quality from the effects produced by high ...

Isolation of Specific Genomic Regions and Identification of Associated Molecules by enChIP

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. To enable the non-biased identification of molecules interacting with a specific genomic region of interest, we recently developed the engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) technique. Here, we describe how to use enChIP to isolate specific genomic regions and identify the associated proteins and RNAs. First, a genomic region of interest is tagged with a transcription activator-like (TAL) protein or a clustered regularly interspaced short palindromic repeats (CRISPR) complex consisting of a catalytically inactive form of Cas9 and a guide RNA. Subsequently, the chromatin is crosslinked and fragmented by sonication. The tagged locus is then immunoprecipitated and the crosslinking is reversed. Finally, the proteins or RNAs that are associated with the isolated chromatin are subjected to mass spectrometric or RNA sequencing analyses, respectively. This approach allows the successful identification of proteins and RNAs associated with a genomic region of interest.

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. This protocol describes procedures to perform engineered DNA-binding molecule-mediated chromatin imunoprecipitation (enChIP) for identification of proteins and RNAs associated with a specific genomic region.

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the ...

Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans

In the last decades, the prevalence of neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD), has grown. These age-associated disorders are characterized by the appearance of protein aggregates with fibrillary structure in the brains of these patients. Exactly why normally soluble proteins undergo an aggregation process remains poorly understood. The discovery that protein aggregation is not limited to disease processes and instead part of the normal aging process enables the study of the molecular and cellular mechanisms that regulate protein aggregation, without using ectopically expressed human disease-associated proteins. Here we describe methodologies to examine inherent protein aggregation in Caenorhabditis elegans through complementary approaches. First, we examine how to grow large numbers of age-synchronized C. elegans to obtain aged animals and we present the biochemical procedures to isolate highly-insoluble-large aggregates. In combination with a targeted genetic knockdown, it is possible to dissect the role of a gene of interest in promoting or preventing age-dependent protein aggregation by using either a comprehensive analysis with quantitative mass spectrometry or a candidate-based analysis with antibodies. These findings are then confirmed by in vivo analysis with transgenic animals expressing fluorescent-tagged aggregation-prone proteins. These methods should help clarify why certain proteins are prone to aggregate with age and ultimately how to keep these proteins fully functional.

Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans

The goal of the method presented here is to explore protein aggregation during normal aging in the model organism C. elegans. The protocol represents a powerful tool to study the highly insoluble large aggregates that form with age and to determine how changes in proteostasis impact protein aggregation.

In the last decades, the prevalence of neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD), has grown. These ...

Sequential Immunofluorescence and Immunohistochemistry on Cryosectioned Zebrafish Embryos

Investigation of intercellular interactions often requires discrete labeling of specific cell populations and precise protein localization. The zebrafish embryo is an excellent tool for examining such interactions with an in vivo model. Whole-mount immunohistochemical and immunofluorescence assays are frequently applied in zebrafish embryos to assess protein expression. However, it can be difficult to achieve accurate mapping of co-localized proteins in three-dimensional space. In addition, some studies may require the use of two antibodies that are not compatible with the same technique (e.g., antibody 1 is only suitable for immunohistochemistry and antibody 2 is only suitable for immunofluorescence). The purpose of the method described herein is to perform sequential immunofluorescence and/or immunohistochemistry on individual cryosections derived from early-stage zebrafish embryos. Here we describe the use of sequential rounds of immunofluorescence, imaging, immunohistochemistry, imaging for a single cryosection in order to achieve precise identification of protein expression at the single-cell level. This methodology is suitable for any study in early-stage zebrafish embryos that requires accurate identification of multiple protein targets in individual cells.

This protocol demonstrates sequential immunofluorescence and immunohistochemistry on cryosections from early-stage zebrafish embryos enabling precise colocalization analyses in specific cell populations.

Investigation of intercellular interactions often requires discrete labeling of specific cell populations and precise protein localization. The zebrafish ...

Visualization of DNA Repair Proteins Interaction by Immunofluorescence

Mammalian cells are constantly exposed to chemicals, radiations, and naturally occurring metabolic by-products, which create specific types of DNA insults. Genotoxic agents can damage the DNA backbone, break it, or modify the chemical nature of individual bases. Following DNA insult, DNA damage response (DDR) pathways are activated and proteins involved in the repair are recruited. A plethora of factors are involved in sensing the type of damage and activating the appropriate repair response. Failure to correctly activate and recruit DDR factors can lead to genomic instability, which underlies many human pathologies including cancer. Studies of DDR proteins can provide insights into genotoxic drug response and cellular mechanisms of drug resistance.
There are two major ways of visualizing&#160;proteins in vivo: direct observation, by tagging the protein of interest with a fluorescent protein and following it by live imaging, or indirect immunofluorescence on fixed samples. While visualization of fluorescently tagged proteins allows precise monitoring over time, direct tagging in N- or C-terminus can interfere with the protein localization or function. Observation of proteins in their unmodified, endogenous version is preferred. When DNA repair proteins are recruited to the DNA insult, their concentration increases locally and they form groups, or &#8220;foci&#8221;, that can be visualized by indirect immunofluorescence using specific antibodies.
Although detection of protein foci does not provide a definitive proof of direct interaction, co-localization of proteins in cells indicates that they regroup to the site of damage and can inform of the sequence of events required for complex formation. Careful analysis of foci spatial overlap in cells expressing wild type or mutant versions of a protein can provide precious clues on functional domains important for DNA repair function. Last, co-localization of proteins indicates possible direct interactions that can be verified by co-immunoprecipitation in cells, or direct pulldown using purified proteins.

Following DNA damage, human cells activate essential repair pathways to restore the integrity of their genome. Here, we describe the method of indirect immunofluorescence as a means to detect DNA repair proteins, analyze their spatial and temporal recruitment, and help interrogate protein-protein interaction at the sites of DNA damage.