This research is funded by the MRC and the BBSRC grants. We thank Agata Zieba and Katerina Pardalis in Ulf Landegren's Lab, Uppsala University for helping with setting up the technique and useful advice. Olink Biosience for technical help and the presentation.

1. Plating of Cells and Treatment with BMP
<ol>
 <li>Culture Neuro2a cells in GMEM supplemented with 10% FBS, 1x Non-Essential amino acids (NEAM), 1x sodium pyruvate and 1x L-glutamine. Split the cells using trypsin (0.05%-EDTA) and plate 15.000-20.000 cells per well in a 16-well chamber slide. 
 
 Culture HEK293T or Cos7 cells in DMEM supplemented with 10% FBS, 1x L-glutamine and 1x Penicillin/Streptomycin. Sterilize polysine slides by immersing them in 70% ethanol and using Immedge pen draw 1 cm2 wells. Split the cells using trypsin (0.05%-EDTA) and plate 20.000-25.000 cells per well in 50 &mu;l medium.</li>
 <li>Incubate the cells at 37&deg;C in a humidified, 5% CO2 incubator.</li>
 <li>After 24 h replace the medium with serum free GMEM (supplemented with 0.1% albumin, 1x NEAM, 1x sodium pyruvate and 1x L-glutamine) for Neuro2a or with serum free DMEM (supplemented with L-Glutamine and 1x Penicillin/Streptomycin) for HEK293T/Cos7 and incubate the cells for 2-3 h. Keep three wells with cells under normal culturing conditions (i.e. 10% FBS) to use as controls.</li>
 <li>Treat the cells with dorsomorphin (inhibitor of the BMP pathway, 2 &mu;M) or BMP4 (25 ng/ml) for the desired time (e.g. for 10 min-1 h) 5.</li>
</ol>
2. Fixation and Permeabilization
<ol>
 <li>Prepare the fixative, 4% PFA. Weigh 4 g of PFA to prepare a 4% solution in 100 ml PBS. Warm the solution at 50&deg;C until it is clear. Filter the solution through a 0.22 &mu;m filter and store at 4&deg;C until use. Use fresh fixative (not stored for more than 2 days).</li>
 <li>Aspirate the medium from the wells and wash with 1xPBS. Do not pipette the solutions directly onto the cells as this may result in detachment of the cells. Before going to the next step, if chamber slides are used remove the chambers, but leave the silicon around the wells.</li>
 <li>Add 50 &mu;l 4% PFA and incubate for 10min, at RT, without agitation. </li>
 <li>Wash the cells with PBS for 3 x 5 min in a Coplin jar with agitation at RT.</li>
 <li>Treat the cells with 0,5% Triton X-100 in PBS for 10 min without agitation at RT.</li>
 <li>Wash the cells with 0,05% Tween 20 in TBS (TBS-T) for 3 x 5 min in a Coplin jar with agitation at RT.</li>
</ol>
3. Blocking
<ol>
 <li>Tap off the TBS-T. Add one drop of Duolink II Blocking Solution (1x) per well.</li>
 <li>Incubate the slides in a pre-heated humidity chamber for 1 h at 37&deg;C.</li>
</ol>
4. Primary Antibodies
<ol>
 <li>Mix and dilute a-P-Smad1/5/8 (rabbit polyclonal) at 1:100 and a-Smad4 (mouse monoclonal) at 1:100 in the Duolink II Antibody Diluent (1x). Prepare also a-P-Smad1/5/8 and a-Smad4 alone at the same concentrations to be used for controls.</li>
 <li>Tap off the Blocking Solution from the slides. Remove as much blocking solution as possible, but take extra care not to let the cells dry before adding the antibody. Add 40 &mu;l of the antibody solutions to the wells. In one well add only Antibody Diluent as an additional negative control.</li>
</ol>
5. PLA probes
<ol>
 <li>Dilute the two PLA probes (Duolink II anti-Mouse MINUS and Duolink II anti-Rabbit PLUS) 1:5 in Antibody Diluent.</li>
 <li>Tap off the primary antibody solution from the slides. Wash the slides 2 times 5 min each with 1x Duolink II Wash Buffer A in a Coplin jar with agitation at RT.</li>
 <li>Tap off the Wash Buffer A from the slides and add the PLA probe solution (40 &mu;l/well). Incubate the slides in a pre-heated humidity chamber for 1 h at 37&deg;C.</li>
</ol>
6. Ligation
<ol>
 <li>Vortex the Duolink II Ligation stock (5x) and dilute 1:5 in high purity water and mix. When calculating the volume of the water, take into account the volume of the Ligase that will be added at a final dilution of 1:40 just before the addition of the mix to the wells.</li>
 <li>Tap off the PLA probe solution from the slides. Wash the slides in 1x Wash Buffer A for 2 times 5 min each in a Coplin jar with agitation at RT.</li>
 <li>Take out the Ligase from the freezer using a freezing block (-20&deg;C). Add the Ligase to the Ligation solution (prepared in 6.1) at a 1:40 dilution and vortex.</li>
 <li>Tap off the Wash Buffer A from the slides and add the Ligation-Ligase solution to each well (40 &mu;l/well). Incubate the slides in a pre-heated humidity chamber for 30 min at 37&deg;C.</li>
</ol>
7. Amplification
Note: Light sensitive reagents. Keep the slides protected from light.
<ol>
 <li>Dilute the Duolink II Amplification stock (5x) 1:5 in high purity water and mix. When calculating the volume of the water take into account the volume of the Polymerase that will be added at a final dilution of 1:80 just before the addition of the mix to the wells.</li>
 <li>Tap off the Ligation-Ligase solution from the slides. Wash the slides in 1x Wash Buffer A with for 2 times 2 min each in a Coplin jar with agitation at RT.</li>
 <li>Take out the Polymerase from the freezer using a freezing block (-20&deg;C). Add the Polymerase to the Amplification solution (prepared in 7.1) at a 1:80 dilution and vortex.</li>
 <li>Tap off the Wash Buffer A from the slides and add the Amplification-Polymerase solution to each well (40 &mu;l/well). Incubate the slides in a pre-heated humidity chamber for 100 min at 37&deg;C.</li>
</ol>
8. Preparation for Imaging
Note: Light sensitive reagents. Keep the slides protected from light.
<ol>
 <li>Tap off the Amplification-Polymerase solution from the slides and wash for 2 times 10min each in 1x Duolink II Wash Buffer B in a Coplin jar with agitation at RT.</li>
 <li>Dip the slides in 0.1 x Wash Buffer B.</li>
 <li>Remove the silicone from the 16-well slide completely. Add ~ 40&mu;l of the Duolink II Mounting Medium with DAPI on the coverslip and gently place it over the samples pressing it slightly so that there are no air bubbles under the cover slip. Fix and seal the coverslip on the slide using nail polish. Wait for at least 15min before proceeding to imaging.</li>
 <li>Analyze the samples with a confocal or fluorescence microscope. Obtain digital images.</li>
</ol>
9. Quantification
Note: Light sensitive reagents. Keep the slides protected from light.
<ol>
 <li>Use Duolink ImageTool to count the signals on the images to obtain a measurement of the signaling level.</li>
 <li>Compare the measurements and make a graph.</li>
</ol>
10. Representative Results
The result from an in situ PLA experiment shows as discrete fluorescent spots. The location of the signal depends on the specific proteins studied. 
<img alt="Figure 1" src="/files/ftp_upload/2631/2631fig1.jpg" /> Figure 1. In situ PLA on Neuro2a cells after BMP4 stimulation. Cells were treated with 2 &mu;M dorsomorphin (inhibitor of the BMP pathway) (A) or 25 ng/ml BMP4 (B) for 60 min or left untreated (C-E). Antibodies against P-Smad1/5/8 and Smad4 were used to detect the active complexes in A and B. The primary antibodies a-P-Smad1/5/8 alone (C) and a-Smad4 alone (D) or omission of the primary antibodies (E) were used as controls. Blue: DAPI; Red: PLA signal. Pictures were obtained with a Leica SP5 confocal microscope.
(F) The PLA signals were counted with the Duolink ImageTool software and the average number of spots in the nucleus per cell is presented in the graph. (G) Neuro2a cells were left untreated or treated with Dorsomorphin (2 &mu;M) or BMP4 (25 ng/ml) for 60 min. The cells were lysed and the proteins were subjected to SDS-PAGE and analysed by immunoblotting with a-P-Smad1/5/8 and a-PCNA as loading control. (*) non-specific band. Note that the a-P-Smad1/5/8 antibody cannot distinguish between the 3 different proteins present in complex with Smad4.

<ol>
	<li>Miyazono, K., Kamiya, Y., Morikawa, M. B. o. n. e. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=morphogenetic+protein+receptors+and+signal+transduction.">morphogenetic protein receptors and signal transduction.</a> J Biochem. 147, 35-51 (2010).</li><li>Gustafsdottir, 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=Proximity+ligation+assays+for+sensitive+and+specific+protein+analyses.">Proximity ligation assays for sensitive and specific protein analyses.</a> Anal Biochem. 345, 2-9 (2005).</li><li>Soderberg, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+observation+of+individual+endogenous+protein+complexes+in+situ+by+proximity+ligation.">Direct observation of individual endogenous protein complexes in situ by proximity ligation.</a> Nat Methods. 3, 995-1000 (2006).</li><li>Soderberg, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterizing+proteins+and+their+interactions+in+cells+and+tissues+using+the+in+situ+proximity+ligation+assay.">Characterizing proteins and their interactions in cells and tissues using the in situ proximity ligation assay.</a> Methods. 45, 227-232 (2008).</li><li>Yu, P. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dorsomorphin+inhibits+BMP+signals+required+for+embryogenesis+and+iron+metabolism.">Dorsomorphin inhibits BMP signals required for embryogenesis and iron metabolism.</a> Nat Chem Biol. 4, 33-41 (2008).</li><li>Baan, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+proximity+ligation+detection+of+c-Jun/AP-1+dimers+reveals+increased+levels+of+c-Jun/Fra1+complexes+in+aggressive+breast+cancer+cell+lines+in+vitro+and+in+vivo.">In situ proximity ligation detection of c-Jun/AP-1 dimers reveals increased levels of c-Jun/Fra1 complexes in aggressive breast cancer cell lines in vitro and in vivo.</a> Mol Cell Proteomics. 9, 1982-1990 .</li><li>Massinen, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+interaction+of+DYX1C1+with+estrogen+receptors+suggests+involvement+of+hormonal+pathways+in+dyslexia.">Functional interaction of DYX1C1 with estrogen receptors suggests involvement of hormonal pathways in dyslexia.</a> Hum Mol Genet. 18, 2802-2812 (2009).</li><li>Thymiakou, E., Zannis, V. I., Kardassis, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physical+and+functional+interactions+between+liver+X+receptor/retinoid+X+receptor+and+Sp1+modulate+the+transcriptional+induction+of+the+human+ATP+binding+cassette+transporter+A1+gene+by+oxysterols+and+retinoids.">Physical and functional interactions between liver X receptor/retinoid X receptor and Sp1 modulate the transcriptional induction of the human ATP binding cassette transporter A1 gene by oxysterols and retinoids.</a> Biochemistry. 46, 11473-11483 (2007).</li></ol>

The production of this video-article was sponsored by OLINK who makes a reagent used in the proximity ligation assay procedure.

The visualization of protein complexes in situ is in great demand, particularly for studies in signaling where protein interaction and protein modification are the means that cells use for sending a signal from their surface to the nucleus. It has not been possible to visualize and quantify complexes between two endogenous proteins in situ with immunofluorescence before. Co-localization of antibodies exhibits low resolution and cannot be used to visualize true interaction. PLA is a new technique that researchers have started using in different systems with great success 6, 7. Here, we demonstrate how to use PLA not only to visualize but also to quantify endogenous complexes between Smad effectors activated downstream of BMP stimulation over time. We used different tissue culture cells including Neuro2a and relied on commercial antibodies raised in different species (mouse a-Smad4 and rabbit a-P-Smad1/5/8) to achieve this (fig 1). This method allowed us, for the first time, to see the activation of BMP effector-complexes over time and not just the presence of the phosphorylated Smad1/5/8, which may not all be engaged in active complexes with Smad4. We counted the signals (fig 1F) and compared the measurement with the quantification obtained from immunoblot of the same cells 8 (figure 1G). We concluded that the number of spots provide an accurate comparative measurement of signaling levels over time. The technique was also applied on other cell lines (HEK293T and Cos7) with similar results (data not shown).
The principle of the technology is based on two unique probes provided with the Duolink kit. Each PLA probe consists of a secondary antibody attached to a unique synthetic oligonucleotide, which acts as a reporter. The proximity of the probes allows DNA ligation at the exact location where these probes are attached in proximity. The distance of the oligonucleotides, which allows DNA hybridization and ligation is small (&lt;40nm) and therefore, only proteins that interact can allow ligation. The ligated DNA can then be amplified and detected with hybridization of labeled oligonucleotides of the amplified sequence. The amplification is specific as it depends on DNA-DNA hybridization principles and also provides high sensitivity as the ligated DNA is amplified several fold resulting in enhancement of the initial ligation event. The amplified DNA is detected by hybridization with labeled oligonucleotides and produces a visible and rather large spot. As the spots can be counted, the PLA signal provides not only the exact spatial information (the location of the interaction events) but also an objective way of quantifying these events 2-4.
Other techniques that are employed for the detection of protein interactions include co-immunoprecipitation, immunofluoresence and Fluorescent Resonance Energy Transfer (FRET). Co-immunoprecipitation assays are widely used enabling the isolation of protein complexes. The method is accompanied by immunoblotting which means that the proteins must be expressed at relatively high levels in order to be detected. Problems of high background or non-specific binding of some proteins to the beads are sometimes difficult to overcome in co-immunoprecipitation. In addition, co-immunoprecipitation assays allow the detection of proteins that can be part of a protein complex but cannot distinguish those that are in very close proximity interacting physically with each other. Furthermore, co-immunoprecipitations cannot provide quantitation in single cell resolution or information on the localization of the protein complexes in the cell. Detection of co-existence of protein complexes in a cell or a cell compartment by immunofluoresence is based on the overlap of diffused signals and therefore, has low spatial resolution and cannot provide accurate information on protein interactions. Quantification of the immunofluoresence signal can be done by visual estimation only if the difference is several fold, and cannot be accurate or measurable as the PLA spots. FRET allows the visualization of protein complexes in live cells and overcomes the need for antibodies and the exposure of the epitope. However, FRET usually depends on the overexpression of artificial fused proteins, which may not reflect the endogenous protein complexes as in the PLA.
Blocking conditions in the PLA protocol are critical as antibody aggregation may occur and increase the background. Alternative blocking conditions can be found in antibody product sheets. Appropriate controls with primary and secondary antibody omission provide information on what may have gone wrong and they need to be always included. It is important to screen different antibodies that will give the least background when they are used alone and the best signal when they are together. Fixation of cells is critical as over-fixing may cause protein complexes to fully denature and degrade, whereas insufficient fixation may lead to loss of protein complexes during the steps of the procedure. This can be controlled by lowering the concentration of the fixative to 3% or by limiting the fixation time. However, the fixation depends also on whether the primary antibodies recognize denatured proteins or not. As the PLA technique is very sensitive, special care is needed to keep the incubation times and micro conditions equal among different samples.
The samples should not be left to dry at any case before the final step. Drying of the samples can lead to increased background. The complete removal of the blocking solution is also critical in order to avoid background. The wash buffers should always be brought to room temperature before use, as low temperature slows the enzymatic reactions. For further troubleshooting refer to the manual of the Kit.
It is useful to use stains to ascertain the sub-cellular localization of the PLA signal (e.g. DAPI, actin etc). Here we have used DAPI (in the mounting medium) to stain the nuclei. PLA is compatible with the use of immunofluorescence with antibodies to determine cell type or cellular compartment (not shown here). Accurate quantification can be done by counting the signals and the use of specific software (Duolink Image Tool, Olink Biosience).
In conclusion we have demonstrated how to use in situ PLA, using the Duolink kit to detect BMP signaling in fixed cells and presented its advantages: easy to use, highly sensitive, no background, and a unique ability to quantify active BMP signaling in situ.
The limitation of applying this technique to study different protein interactions depends on the availability and quality of the primary antibodies that recognize the proteins under investigation.

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>GMEM</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>21710025</td>
<td>&nbsp;</td>
<tr>
<td>DMEM</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>21063029</td>
<td>&nbsp;</td>
<tr>
<td>FBS</td>
<td>&nbsp;</td>
<td>AutogenBioclear</td>
<td>7.01</td>
<td>&nbsp;</td>
<tr>
<td>Non-essential Amino Acids</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11149068</td>
<td>&nbsp;</td>
<tr>
<td>Sodium Pyruvate</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11360070</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin / Streptomycin</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15140122</td>
<td>&nbsp;</td>
<tr>
<td>L-glutamine</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>25030-054</td>
<td>&nbsp;</td>
<tr>
<td>Trypsin, 0.05% (1X) with EDTA 4Na, liquid</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>25300-054</td>
<td>&nbsp;</td>
<tr>
<td>16-well chamber slides</td>
<td>&nbsp;</td>
<td>Lab-Tek</td>
<td>178599</td>
<td>&nbsp;</td>
<tr>
<td>Polysine</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>631-0107</td>
<td>&nbsp;</td>
<tr>
<td>Cover glass</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>631-0138</td>
<td>&nbsp;</td>
<tr>
<td>a-pSmad1/5/8</td>
<td>&nbsp;</td>
<td>Cell Signaling</td>
<td>9511</td>
<td>&nbsp;</td>
<tr>
<td>a-Smad4 (B8) </td>
<td>&nbsp;</td>
<td>Santa Cruz Biotechnology</td>
<td>sc-7966</td>
<td>&nbsp;</td>
<tr>
<td>Dorsomorphin</td>
<td>&nbsp;</td>
<td>Merck</td>
<td>171260-10MG</td>
<td>&nbsp;</td>
<tr>
<td>Recombinant Mouse BMP-4</td>
<td>&nbsp;</td>
<td>R&amp;D</td>
<td>5020-BP-010</td>
<td>&nbsp;</td>
<tr>
<td>PFA</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>P6148</td>
<td>&nbsp;</td>
<tr>
<td>Triton-X 100</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>T8787</td>
<td>&nbsp;</td>
<tr>
<td>Tween-20</td>
<td>&nbsp;</td>
<td>Promega</td>
<td>H5151</td>
<td>&nbsp;</td>
<tr>
<td><a href="http://www.olink.webbhuset.com/products_services/duolink_products.php?menu=380">Duolink II Detection Reagents (Orange)</a></td>
<td>&nbsp;</td>
<td>OLINK</td>
<td>92007-0100</td>
<td>&nbsp;</td>
<tr>
<td><a href="http://www.olink.webbhuset.com/products_services/duolink_products.php?menu=380">Duolink II PLA probe anti-Mouse MINUS</a></td>
<td>&nbsp;</td>
<td>Olink Bioscience</td>
<td>92004-0100</td>
<td>&nbsp;</td>
<tr>
<td><a href="http://www.olink.webbhuset.com/products_services/duolink_products.php?menu=380">Duolink II PLA probe anti-Rabbit PLUS</a></td>
<td>&nbsp;</td>
<td>Olink Bioscience</td>
<td>92002-0100</td>
<td>&nbsp;</td>
<tr>
<td><a href="http://www.olink.webbhuset.com/products_services/duolink_products.php?menu=380">Duolink II Mounting Medium with DAPI</a></td>
<td>&nbsp;</td>
<td>Olink Bioscience</td>
<td>82040-0005</td>
<td>&nbsp;</td>
<tr>
<td><a href="http://www.olink.webbhuset.com/products_services/duolink_products.php?menu=380">Duolink II Wash Buffers Fluorescence</a></td>
<td>&nbsp;</td>
<td>Olink Bioscience</td>
<td>82049</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

detection of signaling effector-complexes downstream of bmp4 using in situ pla, a proximity ligation assay

BMPs are responsible for a wide range of developmental and biological effects. BMP receptors activate (phosphorylate) the Smad1/5/8 effectors, which then, form a complex with Smad4 and translocate to the nucleus where they function as transcription factors to initiate BMP specific downstream effects 1. Traditional immuno-fluorescence techniques with antibodies against phospho-Smad peptides exhibit low sensitivity, high background and offer gross quantification as they rely on intensity of the antibody signal particularly if this is photosensitive fluorescent. In addition, phospho-Smads may not all be in complex with Smad4 and engaged in active transcription.
In situ PLA is a technology capable of detecting protein interactions with high specificity and sensitivity 2-4. This new technology couples antibody recognition with the amplification of DNA surrogate of the protein. It generates a localized, discrete signal in a form of spots revealing the exact position of the recognition event. The number of signals can be counted and compared providing a measurement. We applied in situ PLA, using the Duolink kit, with a combination of antibodies that allows the detection of the BMP signaling effectors phospho-Smad1/5/8 and Smad4 only when these are in proximity i.e. in a complex, which occurs only with signaling activation. This allowed for the first time, the visualization and measurement of endogenous BMP signaling with high specificity and sensitivity in a time course experiment under BMP4 stimulation.

Watch this Scientific Journal Video about Detection of Signaling Effector-Complexes Downstream of BMP4 Using in situ PLA, a Proximity Ligation Assay at JoVE.com

Detection of Signaling Effector-Complexes Downstream of BMP4 Using in situ PLA, a Proximity Ligation Assay

Here we show how to use Proximity Ligation Assay (PLA), with a combination of antibodies to visualize Bone Morphogenetic Protein (BMP) signaling in fixed cells. This technique allowed us to follow the nuclear accumulation of endogenous BMP activated effector-complexes and quantify their levels over time under BMP4 stimulation.

Detection of Signaling Effector-Complexes Downstream of BMP4 Using in situ PLA, a Proximity Ligation Assay

BMPs are responsible for a wide range of developmental and biological effects. BMP receptors activate (phosphorylate) the Smad1/5/8 effectors, which then, ...

detection-signaling-effector-complexes-downstream-bmp4-using-situ-pla

Research

JoVE Journal

Biology

31.7K Views.  Imperial College, Hammersmith Hospital. Here we show how to use Proximity Ligation Assay (PLA), with a combination of antibodies to visualize Bone Morphogenetic Protein (BMP) signaling in fixed cells. This technique allowed us to follow the nuclear accumulation of endogenous BMP activated effector-complexes and quantify their levels over time under BMP4 stimulation.

Video: Detection of Signaling Effector-Complexes Downstream of BMP4 Using in situ PLA, a Proximity Ligation Assay

Double Whole Mount in situ Hybridization of Early Chick Embryos

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  gene expression in brain, neural tube, somite and heart primordia formation. This video demonstrates the different steps in 2-color whole mount in situ hybridization; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, the embryo is processed for prehybridization. The embryo is then hybridized with two different probes, one coupled to DIG, and one coupled to FITC.  Following overnight hybridization, the embryo is incubated with DIG coupled antibody. Color reaction for DIG substrate is performed, and the region of interest appears blue. The embryo is then incubated with FITC coupled antibody. The embryo is processed for color reaction with FITC, and the region of interest appears red. Finally, the embryo is fixed and processed for phtograph and sectioning. A troubleshooting guide is also presented.

This video demonstrates 2-color whole mount in situ hybridization, a method by which the spatial and temporal expression pattern of 2 different genes can be visualized in young chick embryos. This method was originally introduced by David Wilkinson, Domingos Henrique, Phil Ingham and David Ish -Horowicz.

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

Rapid Homogeneous Detection of Biological Assays Using Magnetic Modulation Biosensing System

A magnetic modulation biosensing system (MMB) [1,2] rapidly and homogeneously detected biological targets at low concentrations without any washing or separation step. When the IL-8 target was present, a 'sandwich'-based assay attached magnetic beads with IL-8 capture antibody to streptavidin coupled fluorescent protein via the IL-8 target and a biotinylated IL-8 antibody. The magnetic beads are maneuvered into oscillatory motion by applying an alternating magnetic field gradient through two electromagnetic poles. The fluorescent proteins, which are attached to the magnetic beads are condensed into the detection area and their movement in and out of an orthogonal laser beam produces a periodic fluorescent signal that is demodulated using synchronous detection. The magnetic modulation biosensing system was previously used to detect the coding sequences of the non-structural Ibaraki virus protein 3 (NS3) complementary DNA (cDNA) [2]. The techniques that are demonstrated in this work for external manipulation and condensation of particles may be used for other applications, e.g. delivery of magnetically-coupled drugs in-vivo or enhancing the contrast for in-vivo imaging applications.

Magnetic modulation biosensing system is utilized to rapidly, sensitively and simply detect biological assays, such as DNA molecules and proteins.

A magnetic modulation biosensing system (MMB) [1,2] rapidly and homogeneously detected biological targets at low concentrations without any washing or ...

Detection of Viral RNA by Fluorescence in situ Hybridization (FISH)

Viruses that infect cells elicit specific changes to normal cell functions which serve to divert energy and resources for viral replication. Many aspects of host cell function are commandeered by viruses, usually by the expression of viral gene products that recruit host cell proteins and machineries. Moreover, viruses engineer specific membrane organelles or tag on to mobile vesicles and motor proteins to target regions of the cell (during de novo infection, viruses co-opt molecular motor proteins to target the nucleus; later, during virus assembly, they will hijack cellular machineries that will help in the assembly of viruses). Less is understood on how viruses, in particular those with RNA genomes, coordinate the intracellular trafficking of both protein and RNA components and how they achieve assembly of infectious particles at specific loci in the cell. The study of RNA localization began in earlier work. Developing lower eukaryotic embryos and neuronal cells provided important biological information, and also underscored the importance of RNA localization in the programming of gene expression cascades. The study in other organisms and cell systems has yielded similar important information. Viruses are obligate parasites and must utilise their host cells to replicate. Thus, it is critical to understand how RNA viruses direct their RNA genomes from the nucleus, through the nuclear pore, through the cytoplasm and on to one of its final destinations, into progeny virus particles 1.
FISH serves as a useful tool to identify changes in steady-state localization of viral RNA. When combined with immunofluorescence (IF) analysis 22, FISH/IF co-analyses will provide information on the co-localization of proteins with the viral RNA3. This analysis therefore provides a good starting point to test for RNA-protein interactions by other biochemical or biophysical tests 4,5, since co-localization by itself is not enough evidence to be certain of an interaction. In studying viral RNA localization using a method like this, abundant information has been gained on both viral and cellular RNA trafficking events 6. For instance, HIV-1 produces RNA in the nucleus of infected cells but the RNA is only translated in the cytoplasm. When one key viral protein is missing (Rev) 7, FISH of the viral RNA has revealed that the block to viral replication is due to the retention of the HIV-1 genomic RNA in the nucleus 8. 
Here, we present the method for visual analysis of viral genomic RNA in situ. The method makes use of a labelled RNA probe. This probe is designed to be complementary to the viral genomic RNA. During the in vitro synthesis of the antisense RNA probe, the ribonucleotide that is modified with digoxigenin (DIG) is included in an in vitro transcription reaction. Once the probe has hybridized to the target mRNA in cells, subsequent antibody labelling steps (Figure 1) will reveal the localization of the mRNA as well as proteins of interest when performing FISH/IF.

Detection of Viral RNA by Fluorescence in situ Hybridization FISH

A fluorescence in situ hybridization (FISH) method was developed to visually detect viral genomic RNA using fluorescence microscopy. A probe is made with specificity to the viral RNA that can then be identified using a combination of hybridization and immunofluorescence techniques. This technique offers the advantage of identifying the localization of the viral RNA or DNA at steady-state, providing information on the control of intracellular virus trafficking events.

Viruses  that infect cells elicit specific changes to normal cell functions which serve  to divert energy and resources for viral replication. Many ...

Rapid Genetic Analysis of Epithelial-Mesenchymal Signaling During Hair Regeneration

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an attractive model system to study organ development and tissue-specific signaling. Although hair follicle development is genetically tractable, fast and reproducible analysis of factors essential for this process remains a challenge. Here we describe a procedure to generate targeted overexpression or shRNA-mediated knockdown of factors using lentivirus in a tissue-specific manner. Using a modified version of a hair regeneration model 5, 6, 11, we can achieve robust gain- or loss-of-function analysis in primary mouse keratinocytes or dermal cells to facilitate study of epithelial-mesenchymal signaling pathways that lead to hair follicle morphogenesis. We describe how to isolate fresh primary mouse keratinocytes and dermal cells, which contain dermal papilla cells and their precursors, deliver lentivirus containing either shRNA or cDNA to one of the cell populations, and combine the cells to generate fully formed hair follicles on the backs of nude mice. This approach allows analysis of tissue-specific factors required to generate hair follicles within three weeks and provides a fast and convenient companion to existing genetic models.

Tissue-specific analysis of a hair follicle regeneration model using lentivirus to mediate gain- or loss-of-function.

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an ...

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial scientific task. Many disease causing genes in humans have a fly homologue, making Drosophila a good model to study signaling pathways involved in the development of different disorders. Additionally, the tractability of Drosophila simplifies genetic screens to aid in identifying novel therapeutic targets that may regulate metabolism. In order to perform such a screen a simple and fast method to identify changes in the metabolic state of flies is necessary. In general, carbon dioxide production is a good indicator of substrate oxidation and energy expenditure providing information about metabolic state. In this protocol we introduce a simple method to measure CO2 output from flies. This technique can potentially aid in the identification of genetic perturbations affecting metabolic rate.

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are among one of the most common diseases in humans. The genetically tractable model organism D. melanogaster can be used to identify novel genes that regulate metabolism. This paper describes a relatively simple method which allows studying the metabolic rate in flies by measuring their CO2 production.

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial ...

Detection and Analysis of DNA Damage in Mouse Skeletal Muscle In Situ Using the TUNEL Method

Terminal deoxynucleotidyl transferase (TdT) deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) is the method of using the TdT enzyme to covalently attach a tagged form of dUTP to 3&#8217; ends of double- and single-stranded DNA breaks in cells. It is a reliable and useful method to detect DNA damage and cell death in situ. This video describes dissection, tissue processing, sectioning, and fluorescence-based TUNEL labeling of mouse skeletal muscle. It also describes a method of semi-automated TUNEL signal quantitation. Inherent normal tissue features and tissue processing conditions affect the ability of the TdT enzyme to efficiently label DNA. Tissue processing may also add undesirable autofluorescence that will interfere with TUNEL signal detection. Therefore, it is important to empirically determine tissue processing and TUNEL labeling methods that will yield the optimal signal-to-noise ratio for subsequent quantitation. The fluorescence-based assay described here provides a way to exclude autofluorescent signal by digital channel subtraction. The TUNEL assay, used with appropriate tissue processing techniques and controls, is a relatively fast, reproducible, quantitative method for detecting apoptosis in tissue. It can be used to confirm DNA damage and apoptosis as pathological mechanisms, to identify affected cell types, and to assess the efficacy of therapeutic treatments in vivo.

Detection and Analysis of DNA Damage in Mouse Skeletal Muscle In Situ Using the TUNEL Method

This video describes dissection, tissue processing, sectioning, and fluorescence-based TUNEL labeling of mouse skeletal muscle. It also describes a method for semi-automated analysis of TUNEL labeling.

Terminal deoxynucleotidyl transferase (TdT) deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) is the method of using the TdT enzyme to covalently ...

A Protocol for Lentiviral Transduction and Downstream Analysis of Intestinal Organoids

Intestinal crypt-villus structures termed organoids, can be kept in sustained culture three dimensionally when supplemented with the appropriate growth factors. Since organoids are highly similar to the original tissue in terms of homeostatic stem cell differentiation, cell polarity and presence of all terminally differentiated cell types known to the adult intestinal epithelium, they serve as an essential resource in experimental research on the epithelium. The possibility to express transgenes or interfering RNA using lentiviral or retroviral vectors in organoids has increased opportunities for functional analysis of the intestinal epithelium and intestinal stem cells, surpassing traditional mouse transgenics in speed and cost. In the current video protocol we show how to utilize transduction of small intestinal organoids with lentiviral vectors illustrated by use of doxycylin inducible transgenes, or IPTG inducible short hairpin RNA for overexpression or gene knockdown. Furthermore, considering organoid culture yields minute cell counts that may even be reduced by experimental treatment, we explain how to process organoids for downstream analysis aimed at quantitative RT-PCR, RNA-microarray and immunohistochemistry. Techniques that enable transgene expression and gene knock down in intestinal organoids contribute to the research potential that these intestinal epithelial structures hold, establishing organoid culture as a new standard in cell culture.

In this video protocol we give a step by step explanation of lentiviral transduction in organoids of primary intestinal epithelium and of processing and downstream analysis of these cultures by quantitative RT-PCR, RNA-microarray and immunohistochemistry.

Intestinal crypt-villus structures termed organoids, can be kept in sustained culture three dimensionally when supplemented with the appropriate growth ...

Detection of miRNA Targets in High-throughput Using the 3'LIFE Assay

Luminescent Identification of Functional Elements in 3&#8217;UTRs (3&#8217;LIFE) allows the rapid identification of targets of specific miRNAs within an array of hundreds of queried 3&#8217;UTRs. Target identification is based on the dual-luciferase assay, which detects binding at the mRNA level by measuring translational output, giving a functional readout of miRNA targeting. 3&#8217;LIFE uses non-proprietary buffers and reagents, and publically available reporter libraries, making genome-wide screens feasible and cost-effective. 3&#8217;LIFE can be performed either in a standard lab setting or scaled up using liquid handling robots and other high-throughput instrumentation. We illustrate the approach using a dataset of human 3&#8217;UTRs cloned in 96-well plates, and two test miRNAs, let-7c and miR-10b. We demonstrate how to perform DNA preparation, transfection, cell culture and luciferase assays in 96-well format, and provide tools for data analysis. In conclusion 3&#39;LIFE is highly reproducible, rapid, systematic, and identifies high confidence targets.

Luminescent identification of functional elements in 3&#8217; untranslated regions (3&#8217;UTRs) (3&#8217;LIFE) is a technique to identify functional regulation in 3&#8217;UTRs by miRNAs or other regulatory factors. This protocol utilizes high-throughput methodology such as 96-well transfection and luciferase assays to screen hundreds of putative interactions for functional repression.

Luminescent Identification of Functional Elements in 3&#8217;UTRs (3&#8217;LIFE) allows the rapid identification of targets of specific miRNAs within an ...

Study of Endoplasmic Reticulum and Mitochondria Interactions by In Situ Proximity Ligation Assay in Fixed Cells

Structural interactions between the endoplasmic reticular (ER) and mitochondrial membranes, in domains known as mitochondria-associated membranes (MAM), are crucial hubs for cellular signaling and cell fate. Particularly, these inter-organelle contact sites allow the transfer of calcium from the ER to mitochondria through the voltage-dependent anion channel (VDAC)/glucose-regulated protein 75 (GRP75)/inositol 1,4,5-triphosphate receptor (IP3R) calcium channeling complex. While this subcellular compartment is under intense investigation in both physiological and pathological conditions, no simple and sensitive method exists to quantify the endogenous amount of ER-mitochondria contact in cells. Similarly, MAMs are highly dynamic structures, and there is no suitable approach to follow modifications of ER-mitochondria interactions without protein overexpression. Here, we report an optimized protocol based on the use of an in situ proximity ligation assay to visualize and quantify endogenous ER-mitochondria interactions in fixed cells by using the close proximity between proteins of the outer mitochondrial membrane (VDAC1) and of the ER membrane (IP3R1) at the MAM interface. Similar in situ proximity ligation experiments can also be performed with the GRP75/IP3R1 and cyclophilin D/IP3R1 pairs of antibodies. This assay provides several advantages over other imaging procedures, as it is highly specific, sensitive, and suitable to multiple-condition testing. Therefore, the use of this in situ proximity ligation assay should be helpful to better understand the physiological regulations of ER-mitochondria interactions, as well as their role in pathological contexts.

Study of Endoplasmic Reticulum and Mitochondria Interactions by In Situ Proximity Ligation Assay in Fixed Cells

Here, we describe a procedure to visualize and quantify with high sensitivity the endogenous interactions between the endoplasmic reticulum and mitochondria in fixed cells. The protocol features an optimized in situ proximity ligation assay targeting the inositol 1,4,5-triphosphate receptor/glucose-regulated protein 75/voltage-dependent anion channel/cyclophilin D complex at the mitochondria-associated membrane interface.

Structural interactions between the endoplasmic reticular (ER) and mitochondrial membranes, in domains known as mitochondria-associated membranes (MAM), ...

Applications of Spatio-temporal Mapping and Particle Analysis Techniques to Quantify Intracellular Ca2+ Signaling In Situ

Ca2+ imaging of isolated cells or specific types of cells within intact tissues often reveals complex patterns of Ca2+ signaling. This activity requires careful and in-depth analyses and quantification to capture as much information about the underlying events as possible. Spatial, temporal and intensity parameters intrinsic to Ca2+ signals such as frequency, duration, propagation, velocity and amplitude may provide some biological information required for intracellular signalling. High-resolution Ca2+ imaging typically results in the acquisition of large data files that are time consuming to process in terms of translating the imaging information into quantifiable data, and this process can be susceptible to human error and bias. Analysis of Ca2+ signals from cells in situ typically relies on simple intensity measurements from arbitrarily selected regions of interest (ROI) within a field of view (FOV). This approach ignores much of the important signaling information contained in the FOV. Thus, in order to maximize recovery of information from such high-resolution recordings obtained with Ca2+dyes or optogenetic Ca2+ imaging, appropriate spatial and temporal analysis of the Ca2+ signals is required. The protocols outlined in this paper will describe how a high volume of data can be obtained from Ca2+ imaging recordings to facilitate more complete analysis and quantification of Ca2+ signals recorded from cells using a combination of spatiotemporal map (STM)-based analysis and particle-based analysis. The protocols also describe how different patterns of Ca2+ signaling observed in different cell populations in situ can be analyzed appropriately. For illustration, the method will examine Ca2+ signaling in a specialized population of cells in the small intestine, interstitial cells of Cajal (ICC), using GECIs.

Applications of Spatio-temporal Mapping and Particle Analysis Techniques to Quantify Intracellular Ca2+ Signaling In Situ

Genetically encoded Ca2+ indicators (GECIs) have radically changed how in situ Ca2+ imaging is performed. To maximize data recovery from such recordings, appropriate analysis of Ca2+ signals is required. The protocols in this paper facilitate the quantification of Ca2+ signals recorded in situ using spatiotemporal mapping and particle-based analysis.

Ca2+ imaging of isolated cells or specific types of cells within intact tissues often reveals complex patterns of Ca2+ signaling. This activity requires ...

Detection of Signaling Effector-Complexes Downstream of BMP4 Using in situ PLA, a Proximity Ligation Assay

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Chapters in this video

Double Whole Mount in situ Hybridization of Early Chick Embryos

Rapid Homogeneous Detection of Biological Assays Using Magnetic Modulation Biosensing System

Detection of Viral RNA by Fluorescence in situ Hybridization (FISH)

Rapid Genetic Analysis of Epithelial-Mesenchymal Signaling During Hair Regeneration

Measurement of Metabolic Rate in Drosophila using Respirometry

Detection and Analysis of DNA Damage in Mouse Skeletal Muscle In Situ Using the TUNEL Method

A Protocol for Lentiviral Transduction and Downstream Analysis of Intestinal Organoids

Detection of miRNA Targets in High-throughput Using the 3'LIFE Assay

Study of Endoplasmic Reticulum and Mitochondria Interactions by In Situ Proximity Ligation Assay in Fixed Cells

Applications of Spatio-temporal Mapping and Particle Analysis Techniques to Quantify Intracellular Ca²⁺ Signaling In Situ