The authors would like to thank Drs. Hilary Clay, Stephen Wilson, Anna Payne-Tobin, and James Smyth for helpful discussions. Microscopy was done at the Cardiovascular Research Institute Imaging Core at the University of California, San Francisco. This work was supported by NIH K08 HL105657 (LDW) and NIH HL65590 (SRC).

NOTE: All experimental procedures were approved by the UCSF Institutional Animal Care and Use Committee.
1. Cryopreservation and Fixation of Embryonic Mouse Hearts.
1.1) Snap-freezing embryonic hearts
<ol>
	<li>Fill a 3.5 cm petri dish and 7 mm cryomolds with Optimal Cutting Temperature (OCT) medium (see Materials Table). In a chemical hood, cool 2-methylbutane in liquid nitrogen.</li>
	<li>Dispense 30 ml of phosphate buffered saline (PBS) into 10 cm petri dishes, 10 ml of PBS into 3.5 cm petri dishes, and place all petri dishes on ice. Prepare one 10 cm dish and several 3.5 cm dishes per pregnant mouse.</li>
	<li>Isolate embryos as previously described15, performing the dissection in ice-cold PBS.</li>
	<li>Briefly, euthanize the pregnant female using CO2 narcosis and cervical dislocation.
	<ol>
		<li>Make an incision in the abdomen, dissect out the uterus by cutting the vessels along the inner curvature of the uterus, and transfer the uterus to a 10 cm petri dish containing ice-cold PBS.</li>
		<li>Cut the uterus in between each embryo and transfer to an individual 3.5 cm Petri dish containing ice-cold PBS. Isolate each embryo as described15.</li>
		<li>Open the pericardial cavity using fine forceps, remove the heart away from the lungs and vasculature by cutting at the aorta, inferior vena cava, and pulmonary veins, and transfer into the 3.5 cm petri dish containing OCT.</li>
		<li>Let the heart equilibrate in OCT for several seconds, then transfer the heart into the 7 mm mold containing OCT. Orient the anterior wall of the heart to the bottom of the mold.</li>
	</ol>
	</li>
	<li>Gently place the mold into liquid nitrogen-cooled 2-methylbutane. Take care not to allow 2-methylbutane liquid to touch the OCT or heart. Freeze until the OCT is solid white, then transfer the mold to an ice bucket containing dry ice. Move on to the next embryo.</li>
	<li>Wrap cryomolds in foil and store at -80 &#176;C until ready for cryosectioning.</li>
</ol>
1.2) Alternative: Paraformaldehyde (PFA) fixing, cryoprotecting, and OCT embedding embryonic hearts
<ol>
	<li>Fill the wells of a 12-well tissue culture plate with 4% PFA in 1x PBS.</li>
	<li>Dissect out the embryonic hearts as described in 1.1.3. Place each heart into a well containing 4% PFA and fix at 4 &#176;C O/N.</li>
	<li>Cryoprotection: using a plastic transfer pipette, move each heart to an 1.5 ml microcentrifuge tube containing 1.5 ml of 15% sucrose in PBS and gently agitate at 4 &#176;C until the heart sinks to the bottom of the tube (several hours to O/N). Transfer each heart to a 1.5 ml microcentrifuge tube containing 1.5ml of 30% sucrose in PBS and gently agitate at 4 &#176;C again until the heart sinks to the bottom of the tube (several hours to O/N).</li>
	<li>Using a plastic transfer pipette, place the cryoprotected heart into OCT and let it equilibrate for several minutes to remove excess sucrose, then transfer the heart into the 7 mm mold containing OCT. Orient the anterior wall of the heart to the bottom of the mold.</li>
	<li>Freeze the heart in OCT by placing the mold in either liquid nitrogen-cooled 2-methylbutane or dry ice.</li>
	<li>Wrap cryomolds in foil and store at -80 &#176;C until ready for cryosectioning.</li>
</ol>
2. Cryosectioning
<ol>
	<li>Set cryostat temperature to -17 &#176;C.</li>
	<li>Place cryomolds into the cryostat chamber and equilibrate to temperature for 15-20 min.</li>
	<li>Invert the cryomold and use gentle pressure to expel the heart block from the mold. Orient the anterior wall of the heart to the top of the molded tissue block.</li>
	<li>Place a large drop of OCT onto the chuck, and mount the heart block onto the OCT drop to freeze onto the chuck. Keep the orientation such that the anterior wall of the heart is furthest from the chuck.</li>
	<li>Load the chuck and mounted heart block onto the cryostat object holder. Adjust so that the angle of the blade is 3-5&#176; relative to the sample.</li>
	<li>Collect 10 &#181;m sections onto microscope slides that have been pre-treated with a positively charged coating (see Materials Table). Allow to dry completely before storing at -80 &#176;C.</li>
</ol>
3. Immunofluorescence
<ol>
	<li>For snap-frozen sections, fix and permeabilize tissue in acetone for 10 min in a fume hood at RT.</li>
	<li>For snap-frozen and PFA-fixed sections, incubate in PBS-0.1% Triton X-100 for 20 min to remove OCT and to permeabilize PFA-fixed sections.</li>
	<li>Block for 45 min in 1x blocking buffer, diluted in PBS.</li>
	<li>If using a primary antibody generated in mouse, incubate in donkey or goat anti-mouse IgG (H+L) monovalent Fab fragment diluted 1:100 in PBS-0.1% Tween 20 for 45 min at RT (see Discussion).</li>
	<li>Incubate in primary antibody or antibodies diluted in 1x blocking buffer for 2 hr at RT or O/N at 4 &#176;C (see Table of Materials/Equipment for specific dilutions).</li>
	<li>Wash sections in 1x PBS three times for 10 min at RT.</li>
	<li>Incubate in Alexa Fluor-conjugated secondary antibody diluted 1:500 in blocking buffer for 2 hr at RT, protected from light.</li>
	<li>Wash sections in 1x PBS three times for 10 min at RT, protected from light.</li>
	<li>Optional: Incubate in Hoechst dye diluted 1:2,000 in PBS at RT (protected from light) to label nuclei, then rinse with PBS.</li>
	<li>Post-fix labeled sections in 1% PFA for 1 min at RT.</li>
	<li>Mount slides in anti-fade medium (with DAPI if nuclei are not already labeled) by placing two drops of medium on each end of the slide and then covering with a coverslip. Seal coverslips with nail polish. Store protected from light at 4 &#176;C until ready to image.</li>
</ol>
4. Confocal Imaging and Image Analysis
<ol>
	<li>Turn on the appropriate laser wavelengths, camera, and confocal microscope including stage mover and z motor. Launch the imaging software program. Use the 405, 488, and 561 laser wavelengths for imaging Hoechst-, Alexa 488-, and Alexa 568-stained sections, respectively. 
	NOTE: See Materials Table for our hardware and software specifications.</li>
	<li>Mount the control slide (coverslip down for an inverted microscope) on the slide stage.</li>
	<li>Using the 4X objective (see Materials Table), find the sample and area of interest. Capture the image to use as a map when imaging at high magnification.</li>
	<li>Remove the slide, making minimal adjustments to the slide stage. Change to the 60x oil immersion objective (see Materials Table), place a small drop of oil on the objective, and replace the slide (coverslip down) onto the slide stage.</li>
	<li>Find sample again. Set the laser power, exposure time, and binning to the desired levels for each channel. 
	NOTE: We generally use laser power of 0.8, camera exposure time of 100 msec, and binning of 2 (see Materials Table for hardware and software specifications); optimal settings need to be empirically determined for each experiment.
	<ol>
		<li>Once optimal settings are determined, use the same settings for all tissue sections within the experiment. Use the intensity histogram to note the optimal intensity range for each channel (this information will be used for analysis).</li>
	</ol>
	</li>
	<li>Generate a z stack using the acquisition function: select the appropriate laser channels, then choose the upper and lower limits of the z stack. Choose a z stack step size that is one-half the value of the optical slice thickness provided by the software. Click &#8220;run&#8221; to collect the images.</li>
	<li>Use Fiji16 or a comparable program for image analysis. Within Fiji, open the z stack file with Custom color mode option and the channels split into separate windows. Open the &#8220;Adjust Brightness/Contrast&#8221; tool from the Image pulldown menu; within each channel, set the optimal histogram intensity range determined in 4.5.1. Apply these channel ranges to all z stacks being analyzed.</li>
	<li>Merge the individual channels into a single composite image using Image-&#62;Color pulldown menu.</li>
	<li>Create a flattened z stack from the composite image using the Image-&#62;Stacks-&#62;z project menu. This image will be significantly brighter than the 3D image; adjust the histogram intensity range for the control sample to avoid oversaturation, and apply the same settings to the experimental flattened z stack.</li>
	<li>To generate a 3D image, first use the Image-&#62;Stacks-&#62;3D project menu 17. Choose either the x-axis or y-axis of rotation. Set the slice spacing as the same number of microns as the z stack step size. Choose the desired total rotation and set the rotation angle increment to 1. Then open the Image J 3D Viewer from the Plugins pulldown menu. Choose the composite image generated in 4.8, display as Volume, and set the Resampling factor to 1 or 2.</li>
</ol>

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The authors have nothing to disclose.

The optimal tissue fixation technique and dilution must be empirically determined for each antibody. In our hands, snap-freezing is superior to PFA fixation for several cardiomyocyte antigens, including s-&#945;-actinin, &#946;-catenin, &#946;1-integrin, tropomyosin, talin (not shown) and N-cadherin; in contrast, PFA fixation yields superior results for focal adhesion kinase (not shown). The protein crosslinks formed by PFA may mask epitopes and limit antibody binding; antigen retrieval may be required in such cases, and methods for antigen retrieval may be found elsewhere20. PFA concentration or length of fixation can be decreased to reduce epitope masking, with optimal conditions empirically determined for each antibody and at each developmental stage. Appropriate negative controls should be used when characterizing a new antibody or cardiac mutant, including pre-immune serum as the primary antibody control and a &#8220;no primary antibody&#8221; control. Use of knockout mice is an ideal negative control but early lethality prevents their use for many of the gene products studied here.
Use of adequate volumes to completely submerge experimental and control slides in the same immunofluorescence blocking, antibody, and wash solutions was important as was gentle rocking of slides during incubation to ensure uniform exposure of the sections to the solutions. This approach minimized technical variability in staining between sections and slides within an experiment. When cost limits the antibody solution volume, use a Pap pen to limit flow of solutions beyond the tissue sections and keep slides in a humidified chamber during long incubations. If a monoclonal mouse primary antibody - and therefore an anti-mouse secondary antibody - is being used, a second blocking step to cover endogenous mouse immunoglobulins will be necessary (step 3.4) to decrease nonspecific background signal.
Appropriate microscopy and image processing techniques are critical to obtaining biologically accurate information21. The intensity histogram indicates the distribution of pixels at each intensity level (0 &#8211; 65535 levels for a 16-bit image) for each color. Background, brightness, and contrast can be adjusted by setting an intensity display range that flanks the histogram peak; to make valid comparisons between conditions, the same settings between control and experimental conditions must be used.
This protocol provides a reliable method to analyze cardiomyocyte maturation and development in the native embryonic mouse heart. While immunofluorescence of cardiomyocyte-specific proteins is often used to mark cardiomyocytes during development, few studies employ techniques that allow high-resolution analysis of myofibril structure and the emergence of intercalated discs and costameres12-14,22,23. This technique can be used for in vivo assessment of mutations that cause developmental heart defects, as a means of identifying changes in cardiomyocyte maturation that may shed light on mechanisms of structural abnormalities.

During development, heart contractions begin soon after cardiomyocytes migrate to the midline and form the linear heart tube1,2. The sarcomere is the basic contractile unit within the cardiomyocyte; this highly organized cytoskeletal structure contains actin filaments anchored to the Z-disc by sarcomeric &#945;-actinin (s-&#945;-actinin) and myosin fibers anchored to the M line (Figure 1). As the cardiomyocyte matures, sarcomeres assemble in series to form myofibrils that extend across the cell. Myofibrils are anchored to the ends of the cardiomyocyte by the intercalated disc, the cell-cell junctional structure that contains a transitional junction with a subset of Z-disc elements such as s-&#945;-actinin3, adherens junction proteins such as N-cadherin and &#946; catenin, gap junction proteins, and desmosomes (Figure 1)4. Along the longitudinal membrane, the Z-discs of peripheral myofibrils also attach to the cell membrane via costameres; these specialized focal adhesions provide an anchor between the myofibril, plasma membrane, and extracellular matrix to provide additional structural support to the cardiomyocyte (Figure 1)4. Early in heart development, cardiomyocytes are arranged in finger-like projections known as trabeculae that protrude into the ventricular space and contain relatively mature myofibrils5. As heart development proceeds, the cardiomyocytes in the sub-epicardial region proliferate to form the compact myocardium that comprises the ventricular walls, but sarcomere and myofibril assembly are delayed as compared with trabecular myocardium5,6.
Models of sarcomere and myofibril assembly come largely from immunofluorescence studies on cultured cardiomyocytes7-10, which are straightforward but lack a three-dimensional environment, blood flow, and contacts with other cardiac cells present in vivo. High-resolution structural studies using immunofluorescence in the mouse embryonic heart are technically challenging, and few studies have explored the emergence of intercalated discs and costameres during mouse cardiac development. The adherens junction protein &#946; catenin appears to localize to intercalated discs by embryonic day (E) 17.511, N-cadherin localizes to linear structures that may represent intercalated discs by E18.512 versus postnatal day 013, and costameres have been detected at E18.514, but these proteins display diffuse and more continuous membrane distribution at earlier developmental time points11-13.
Here, we describe a straightforward and reproducible method for immunostaining and fluorescence microscopy of sectioned mouse embryonic hearts that allows for detailed analysis of myofibril and cardiomyocyte development, including the emergence of intercalated discs as early as E12.5 and nascent costameres at E16.5. This protocol may be useful for probing the effects of mutations on sarcomere formation as well as myofibril and cardiomyocyte maturation.

<table><tbody><tr><td>Cryomold, Disposable Base Mold, 7x 7 mm</td><td>Richard-Allen Scientific</td><td>58949</td><td>This size not available from Fisher Scientific</td></tr><tr><td>Cryomold, Disposable Base Mold, 15 x 15 mm</td><td>Fisher Scientific</td><td>22-050-159</td><td>Also available from Richard-Allen Scientific, catalog number 58950</td></tr><tr><td>Tissue-Tek Optimal Cutting Temperature (OCT) medium 4583</td><td>VWR</td><td>25608-930</td><td></td></tr><tr><td>2-methyl butane</td><td>Sigma</td><td>M32631</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Fisher Scientific</td><td>T353-500</td><td>Use fresh 4% solution in 1x PBS, pH 7.2-7.4.&amp;nbsp;</td></tr><tr><td>Sucrose</td><td>Sigma</td><td>S0389</td><td>Use 15% and 30% solutions in 1X PBS.</td></tr><tr><td>Disposable transfer pipette</td><td>Fisher Scientific</td><td>S30467-1</td><td></td></tr><tr><td>Superfrost Plus slides</td><td>Fisher Scientific</td><td>12-550-15</td><td>&quot;Plus&quot; indicates positively-charged coating and is critical for maintaining tissue adherence to the slide.</td></tr><tr><td>Acetone</td><td>Sigma</td><td>179124</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>X100</td><td></td></tr><tr><td>Western Blocking Agent</td><td>Roche</td><td>11921673001</td><td>Blocking buffer for immunofluorescence, when secondary antibodies are from different species. Dilute to 1x in PBS.&amp;nbsp;</td></tr><tr><td>Tween 20</td><td>Sigma</td><td>P9416</td><td></td></tr><tr><td>Donkey Anti-mouse IgG (H+L) monovalent Fab fragment</td><td>Jackson ImmunoResearch</td><td>715-007-003</td><td>Goat Anti-mouse IgG (H+L) also available. For blocking endogenous immunoglobulins.</td></tr><tr><td>Anti-s-&amp;#945;-actinin antibody</td><td>Sigma</td><td>A7811</td><td>Mouse monoclonal, clone EA53. Dilute 1:400 for snap-frozen/acetone-fixed sections, dilute 1:300 for PFA-fixed sections. Before incubation on mouse tissue, block endogenous mouse immunoglobulins with anti-mouse IgG (H+L) monovalent Fab fragment.</td></tr><tr><td>Anti-&amp;#946;catenin antibody</td><td>Cell Signaling</td><td>9587s</td><td>Rabbit polyclonal. Requires snap-freezing/acetone fixation. Dilute 1:400.</td></tr><tr><td>Anti-&amp;#946;1 integrin antibody</td><td>R&amp;amp;D</td><td>AF2405</td><td>Goat polyclonal. Dilute 1:200. Snap-frozen/acetone-fixed tissue staining is superior to PFA-fixed tissue.</td></tr><tr><td>Anti-tropomyosin antibody</td><td>Developmental Studies Hybridoma Bank, University of Iowa</td><td>CH1</td><td>Mouse monoclonal. Requires snap-freezing/acetone fixation. Dilute 1:200. Before incubation on mouse tissue, block endogenous mouse immunoglobulins with anti-mouse IgG (H+L) monovalent Fab fragment.</td></tr><tr><td>Anti-N-cadherin antibody</td><td>Santa Cruz</td><td>sc-7939</td><td>Rabbit polyclonal. Dilute 1:200. Snap-frozen/acetone-fixed tissue staining is superior to PFA-fixed tissue.</td></tr><tr><td>Anti-talin antibody</td><td>Sigma</td><td>T3287</td><td>Mouse monoclonal, clone 8d4. Requires snap-freezing/acetone fixation. Dilute 1:200. Before incubation on mouse tissue, block endogenous mouse immunoglobulins with anti-mouse IgG (H+L) monovalent Fab fragment.</td></tr><tr><td>Anti-phosphotyrosine 397 focal adhesion kinase antibody</td><td>Invitrogen</td><td>700255</td><td>Rabbit monoclonal antibody. Requires PFA fixation. Dilute 1:500.</td></tr><tr><td>Alexa Fluor 488 Donkey Anti-Mouse IgG (H+L) Antibody</td><td>Life Technologies</td><td>A-21202</td><td></td></tr><tr><td>Alexa Fluor 568 Donkey Anti-Rabbit IgG Antibody</td><td>Life Technologies</td><td>A10042</td><td></td></tr><tr><td>Alexa Fluor 488 Goat Anti-Mouse IgG (H+L) Antibody</td><td>Life Technologies</td><td>A-11001</td><td></td></tr><tr><td>Alexa Fluor 568 Goat Anti-Rabbit IgG (H+L)Antibody</td><td>Life Technologies</td><td>A-11011</td><td></td></tr><tr><td>Hoechst 33342 dye</td><td>Life Technologies</td><td>H3570</td><td>Nuclear stain</td></tr><tr><td>Vectashield</td><td>Vector labs</td><td>H-1000</td><td></td></tr><tr><td>Coverslips</td><td>Fisher Scientific</td><td>12-548-5M</td><td></td></tr><tr><td>MLC 400B Monolithic Laser Combiner Laser box</td><td>Keysight (formerly Agilent)</td><td></td><td></td></tr><tr><td>Clara Interline CCD camera</td><td>Andor</td><td></td><td></td></tr><tr><td>Eclipse Ti inverted microscope</td><td>Nikon</td><td></td><td></td></tr><tr><td>CSU-X1 Spinning disk confocal scanner unit</td><td>Yokogawa</td><td></td><td></td></tr><tr><td>CFI Plan Apochromat 4X air objective</td><td>Nikon&amp;nbsp;</td><td>Numerical aperture (NA) 0.2, Working distance (WD) 15.7 mm</td><td></td></tr><tr><td>CFI Plan Apochromat VC 60x oil immesion objective (MRD01602)</td><td>Nikon&amp;nbsp;&amp;nbsp;</td><td>NA 1.4, WD 0.13 mm</td><td></td></tr><tr><td>NIS Elements Imaging Software</td><td>Nikon</td><td>http://nikon.com/products/instruments/lineup/bioscience/img_soft/index.htm</td><td></td></tr><tr><td>Image analysis software</td><td>Fiji</td><td>http://fiji.sc/Fiji</td><td></td></tr></tbody></table>

analysis of cardiomyocyte development using immunofluorescence in embryonic mouse heart

During heart development, the generation of myocardial-specific structural and functional units including sarcomeres, contractile myofibrils, intercalated discs, and costameres requires the coordinated assembly of multiple components in time and space. Disruption in assembly of these components leads to developmental heart defects. Immunofluorescent staining techniques are used commonly in cultured cardiomyocytes to probe myofibril maturation, but this ex vivo approach is limited by the extent to which myocytes will fully differentiate in culture, lack of normal in vivo mechanical inputs, and absence of endocardial cues. Application of immunofluorescence techniques to the study of developing mouse heart is desirable but more technically challenging, and methods often lack sufficient sensitivity and resolution to visualize sarcomeres in the early stages of heart development. Here, we describe a robust and reproducible method to co-immunostain multiple proteins or to co-visualize a fluorescent protein with immunofluorescent staining in the embryonic mouse heart and use this method to analyze developing myofibrils, intercalated discs, and costameres. This method can be further applied to assess cardiomyocyte structural changes caused by mutations that lead to developmental heart defects.

Figures 2 through 6 show typical results for co-staining of different proteins in a snap-frozen and acetone-fixed heart. The antibody against s-&#945;-actinin reproducibly labeled Z-discs and intercalated discs with high specificity and minimal background (Figures 2A, 3A, 4A, 5A, 6A, and 6C); Figure 6 demonstrates that the anti-mouse IgG (H+L) monovalent Fab fragment effectively blocks endogenous mouse IgG binding by anti-mouse secondary antibodies. The antibody against adherens junction protein &#946; catenin bound the membrane of both cardiomyocytes and non-cardiomyocyte cells, and co-localization with s-&#945;-actinin occurred in presumed intercalated discs at E16.5 (Figure 2C and D), as expected from the &#946; catenin staining pattern in the adult heart18. &#946;1 integrin immunofluorescence in the embryonic heart is especially challenging and often fails to identify focal adhesions14, but &#946;1 integrin staining in these studies revealed signal with the same periodicity as s-&#945;-actinin-labeled Z-discs, possibly reflecting nascent costameres forming at E16.5 (Figure 3D).
At E12.5, s-&#945;-actinin and tropomyosin (sarcomere thin filament protein) immunofluorescence revealed a staining pattern with regular periodicity in trabecular cardiomyocytes consistent with mature myofibrils in these cells (Figures 4A and 5A for s-&#945;-actinin; Figure 4B for tropomyosin). N-cadherin staining in trabecular cardiomyocytes at E12.5 hearts tended to colocalize with areas of intense s-&#945;-actinin staining (Figure 5B-D and Figure 6A-C) possibly representing intercalated discs. In contrast to trabecular myocytes, s-&#945;-actinin in the compact zone was more punctate than linear, and tropomyosin staining was diffuse rather than linear (Figure 4A and 4B). Thus, sarcomere assembly may occur later in compact compared to trabecular myocardium. Furthermore, differential patterns of s-&#945;-actinin and tropomyosin in the compact zone suggest that s-&#945;-actinin organizes into puncta and immature Z-discs early, while tropomyosin incorporation into the thin filament may be a later event in myofibril assembly.
Figure 7, Movie 1, and Movie 2 demonstrate typical results from a PFA-fixed E12.5 embryonic heart. In these examples, a LifeAct-RFPruby transgenic embryo was used for imaging; the LifeAct-RFPruby transgene19 labels filamentous actin but requires PFA fixation. Z-discs labeled with s-&#945;-actinin were easy to visualize in most areas, but the signal-to-noise ratio was decreased compared to snap-frozen heart sections (Figure 7A); this signal was typical for s-&#945;-actinin immunofluorescence in PFA-fixed tissue, in which epitopes may be masked by protein cross-links. Figure 7B shows co-visualization of filamentous actin and immunolabeled s-&#945;-actinin within myofibrils (arrowheads) and filamentous actin within endocardial cells adjacent to trabecular myocytes (arrows). Three-dimensional image reconstruction revealed additional details: individual cardiomyocytes were more easily discerned, myofibrils within a cardiomyocyte were roughly parallel to one another, but individual cardiomyocytes were oriented at varying angles to one another (Figure 7C and D, Movie 1, and Movie 2). The close approximation between endocardial cells and cardiomyocytes was better appreciated in the three-dimensional views as well.
<img alt="Figure 1" src="/files/ftp_upload/52644/52644fig1.jpg" /> 
Figure 1.&#160;Cardiomyocyte sarcomeres, intercalated discs, and costameres. The Z-disc anchors actin filaments, while the M line anchors myosin fibers, which overlap the actin filaments. The sarcomere comprises one Z-disc &#8211; M line &#8211; Z-disc unit. Multiple sarcomeres in series create a myofibril. The lateral end of the myofibril inserts into the transverse border of the cardiomyocyte at a specialized cell-cell junctional structure called the intercalated disc. Peripheral myofibrils connect to the longitudinal cardiomyocyte plasma membrane via costameres, which form focal adhesions with the extracellular matrix between cardiomyocytes. <a href="https://www.jove.com/files/ftp_upload/52644/52644fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/52644/52644fig2.jpg" /> 
Figure 2.&#160;s-&#945;-actinin and &#946; catenin immunofluorescence at embryonic day 16.5. The heart was excised, snap frozen, cryosectioned, acetone-fixed, and immunostained using (A) mouse monoclonal clone EA53 antibody against s-&#945;-actinin, which labeled cardiomyocyte Z-disc and intercalated discs, and (B) rabbit polycloncal antibody against the adherens junction protein &#946; catenin. (C) Merged images show s-&#945;-actinin and &#946; catenin staining. (D) Magnified area of interest from panel C; asterisks mark presumed intercalated discs with co-localization of s-&#945;-actinin and &#946; catenin. Images were obtained from the peripheral left ventricular wall or compact myocardium, with the epicardial layer at the upper left of panels A-C. Intensity histogram display range 460-1600 (out of possible 0-65535) for both the s-&#945;-actinin/488 nm and &#946; catenin/561 nm laser channels. Scale bar 10 &#181;m. <a href="https://www.jove.com/files/ftp_upload/52644/52644fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/52644/52644fig3.jpg" /> 
Figure 3.&#160;s-&#945;-actinin and &#946;1 integrin immunofluorescence at embryonic day 16.5. The heart was excised, snap frozen, cryosectioned, acetone-fixed, and immunostained using (A) mouse monoclonal clone EA53 antibody against s-&#945;-actinin and (B) goat polyclonal antibody against the focal adhesion protein &#946;1 integrin. (C) Merged images show &#946;1 integrin in cardiomyocyte as well as non-cardiomyocyte cells. Note both diffuse and punctate &#946;1 integrin signal in cardiomyocytes. (D) Magnified area of interest from panel C. Note punctate, periodic &#946;1 integrin staining (arrows) with periodicity similar to nearby s-&#945;-actinin-staining in Z-discs; these structures may represent costameres. Images were obtained from the left ventricular compact myocardium. Intensity histogram display range 460-1200 (out of possible 0-65535) for the s-&#945;-actinin/488 nm laser channel and 460-600 for the &#946;1 integrin/561 nm laser channel. Scale bar 10 &#181;m. <a href="https://www.jove.com/files/ftp_upload/52644/52644fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/52644/52644fig4.jpg" /> 
Figure 4.&#160;s-&#945;-actinin and tropomyosin immunofluorescence at embryonic day 12.5: myofibril organization in trabecular and compact myocardium. Hearts from littermate embryos were excised, snap frozen, cryosectioned, acetone-fixed, and immunostained using (A) mouse monoclonal clone EA53 antibody against s-&#945;-actinin and (B) mouse monoclonal antibody against the myofibril thin filament protein tropomyosin (Developmental Studies Hybridoma Bank CH1). Trabecular (arrows) and compact myocardium (arrowheads) are indicated. Note linear s-&#945;-actinin staining with regular periodicity in the trabecular myocardium, compared to a range of staining patterns including puncta as well as linear staining in the compact layer (A). Note also linear tropomyosin staining with regular periodicity in the trabecular myocardium but more diffuse staining in compact myocardium. Intensity histogram display range 460-1,400 (out of possible 0-65535) for the s-&#945;-actinin channel and 460-1,000 for the tropomyosin channel. Scale bar 10 &#181;m. <a href="https://www.jove.com/files/ftp_upload/52644/52644fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/52644/52644fig5.jpg" /> 
Figure 5.&#160;s-&#945;-actinin and N-cadherin immunofluorescence at embryonic day 12.5: myofibrils and intercalated discs in trabecular cardiomyocytes. The heart was excised, snap frozen, cryosectioned, acetone-fixed, and immunostained using (A) mouse monoclonal clone EA53 antibody against s-&#945;-actinin and (B) rabbit polyclonal antibody against the focal adhesion protein N-cadherin. 0.2 &#181;m optical slices were collected as a z stack, and z stacks were flattened to generate the images. (C) Merged flattened stacks show both N-cadherin and s-&#945;-actinin staining within trabecular cardiomyocytes as well as nuclei labeled with Hoechst dye. (D) Magnified area of interest from panel C; asterisks mark intercalated discs with co-localization of s-&#945;-actinin and N-cadherin. Intensity histogram display range 470-1,200 (out of possible 0-65535) for the Hoechst/405 nm laser channel and 470-2,000 for both the s-&#945;-actinin/488 nm and N-cadherin/561 nm laser channels. Scale bar 10 &#181;m. <a href="https://www.jove.com/files/ftp_upload/52644/52644fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" src="/files/ftp_upload/52644/52644fig6.jpg" /> 
Figure 6.&#160;Anti-mouse IgG (H+L) monovalent Fab fragment effectively blocks endogenous mouse IgG binding by anti-mouse secondary antibodies. The E12.5 embryonic heart was excised, snap frozen, cryosectioned, acetone-fixed, and immunostained. (A-C) Sections were blocked with 1x blocking buffer followed by anti-mouse IgG monovalent Fab fragment, exposed to mouse monoclonal clone EA53 primary antibody against s-&#945;-actinin and rabbit polyclonal primary antibody against N-cadherin, washed, and exposed to Alexa Fluor 488 anti-mouse and Alexa Fluor 586 anti-rabbit secondary antibodies. (A) Merged image using intensity histogram display range 480-2500 (out of possible 0-65535). Asterisks note regions in which N-cadherin signal is restricted to the transverse end of trabecular cardiomyocytes, which likely represents nascent intercalated discs. (B) N-cadherin-only channel using intensity histogram display range 480-2,500. (C) s-&#945;-actinin-only channel using intensity histogram display range 480-2,500. (D-G) Sections were blocked with 1x blocking buffer only (no anti-mouse IgG monovalent Fab fragment blocking step), exposed to rabbit polyclonal primary antibody against N-Cadherin only (no mouse monoclonal primary antibody), washed, and exposed to Alexa Fluor 488 anti-mouse and Alexa Fluor 586 anti-rabbit secondary antibodies. (D) Merged image using intensity histogram display range 480-2500. (E) N-cadherin-only channel using intensity histogram display range 480-2500. (F) s-&#945;-actinin-only channel using intensity histogram display range 480-2,500. (G) s-&#945;-actinin-only channel using high-sensitivity intensity histogram display range 480-530, which reveals background detection of endogenous mouse IgG in the absence of the anti-mouse IgG monovalent Fab fragment blocking step. (H-K) Sections were blocked with 1x blocking buffer followed by anti-mouse IgG monovalent Fab fragment, exposed to rabbit polyclonal primary antibody against N-Cadherin (no mouse monoclonal primary antibody), washed, and exposed to Alexa Fluor 488 anti-mouse and Alexa Fluor 586 anti-rabbit secondary antibodies. (H) Merged image using intensity histogram display range 480-2,500. (I) N-cadherin-only channel using intensity histogram display range 480-2,500. (J) s-&#945;-actinin-only channel using intensity histogram display range 480-2,500. (K) s-&#945;-actinin-only channel using high-sensitivity intensity histogram display range 480-530, which demonstrates the lack of background endogenous mouse IgG detection when the anti-mouse IgG monovalent Fab fragment blocking step is used. <a href="https://www.jove.com/files/ftp_upload/52644/52644fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" src="/files/ftp_upload/52644/52644fig7.jpg" /> 
Figure 7.&#160;s-&#945;-actinin and actin organization in trabecular cardiomyocytes at embryonic day 12.5. The LifeAct-RFPruby transgenic mouse line was used to visualize filamentous actin19, while the mouse monoclonal clone EA53 antibody against s-&#945;-actinin was used to label Z-discs and intercalated discs. Embryos were PFA-fixed. 0.2 &#181;m optical slices were collected as a z stack. (A) Flattened z stack shows that s-&#945;-actinin staining was more diffuse in PFA-fixed tissue than in snap-frozen and acetone fixed sections (Figures 2-5). (B) Flattened z stack shows both filamentous actin and s-&#945;-actinin. Filamentous actin fluorescence localized between Z-discs within myofibrils (arrowheads). Filamentous actin fluorescence was also seen in endocardial cells that line the trabecular myocytes (arrows). (C) Three-dimensional view of the trabecular cardiomyocytes, as viewed from the top of the stack. (D) Three-dimensional view of the trabecular cardiomyocytes, as viewed from the bottom of the stack. Intensity histogram display range 470-900 (out of possible 0-65535) for both the 488 nm laser channel and for the 561 nm laser channel in A and B; display range 460-800 for both channels in C and D. Scale bar 10 &#181;m. <a href="https://www.jove.com/files/ftp_upload/52644/52644fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<a href="https://www.jove.com/files/ftp_upload/52644/Movie1.mov">Movie</a> 1.&#160;360&#176; rotational 3D view of s-&#945;-actinin and actin organization in trabecular cardiomyocytes at embryonic day 12.5. The image stack from Figure 6 was rendered in three dimensions using the Image J 3D Viewer plugin within the Fiji image analysis program. Intensity histogram display range 470-800 (out of possible 0-65535) for both the 488 nm and 561 nm laser channels.
<a href="https://www.jove.com/files/ftp_upload/52644/Movie2.mov">Movie</a> 2.&#160;Selected 3D views of s-&#945;-actinin and actin organization in trabecular cardiomyocytes at embryonic day 12.5. The image stack from Figure 6 was rendered in three dimensions using the Image J 3D Viewer plugin within the Fiji image analysis program. Small rotations around the x, y, and z axes showed relatively aligned myofibrils within cardiomyocytes but poor alignment between most cardiomyocytes. Small rotations also demonstrated the close approximation of endocardial cells lacking s-&#945;-actinin around cardiomyocytes. Intensity histogram display range 470-800 (out of possible 0-65535) for both the 488 nm and 561 nm laser channels.

Watch this Scientific Journal Video about Analysis of Cardiomyocyte Development using Immunofluorescence in Embryonic Mouse Heart at JoVE.com

Analysis of Cardiomyocyte Development using Immunofluorescence in Embryonic Mouse Heart

Mutations that lead to congenital heart defects benefit from in vivo investigation of cardiac structure during development, but high-resolution structural studies in the mouse embryonic heart are technically challenging. Here we present a robust immunofluorescence and image analysis method to assess cardiomyocyte-specific structures in the developing mouse heart.

During heart development, the generation of myocardial-specific structural and functional units including sarcomeres, contractile myofibrils, intercalated ...

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Developmental Biology

21.2K Views.  Northwestern University. Mutations that lead to congenital heart defects benefit from in vivo investigation of cardiac structure during development, but high-resolution structural studies in the mouse embryonic heart are technically challenging. Here we present a robust immunofluorescence and image analysis method to assess cardiomyocyte-specific structures in the developing mouse heart.

Video: Analysis of Cardiomyocyte Development using Immunofluorescence in Embryonic Mouse Heart

Imaging Cleared Embryonic and Postnatal Hearts at Single-cell Resolution

Single clonal tracing and analysis at the whole-heart level can determine cardiac progenitor cell behavior and differentiation during cardiac development, and allow for the study of the cellular and molecular basis of normal and abnormal cardiac morphogenesis. Recent emerging technologies of retrospective single clonal analyses make the study of cardiac morphogenesis at single cell resolution feasible. However, tissue opacity and light scattering of the heart as imaging depth is increased hinder whole-heart imaging at single cell resolution. To overcome these obstacles, a whole-embryo clearing system that can render the heart highly transparent for both illumination and detection must be developed. Fortunately, in the last several years, many methodologies for whole-organism clearing systems such as CLARITY, Scale, SeeDB, ClearT, 3DISCO, CUBIC, DBE, BABB and PACT have been reported. This lab is interested in the cellular and molecular mechanisms of cardiac morphogenesis. Recently, we established single cell lineage tracing via the ROSA26-CreERT2; ROSA26-Confetti system to sparsely label cells during cardiac development. We adapted several whole embryo-clearing methodologies including Scale and CUBIC (clear, unobstructed brain imaging cocktails and computational analysis) to clear the embryo in combination with whole mount staining to image single clones inside the heart. The heart was successfully imaged at single cell resolution. We found that Scale can clear the embryonic heart, but cannot effectively clear the postnatal heart, while CUBIC can clear the postnatal heart, but damages the embryonic heart by dissolving the tissue. The methods described here will permit the study of gene function at a single clone resolution during cardiac morphogenesis, which, in turn, can reveal the cellular and molecular basis of congenital heart defects.

We describe a protocol to volumetrically image fluorescent protein labeled cells deep inside intact embryonic and postnatal hearts. Utilizing tissue-clearing methods in combination with whole mount staining, single fluorescent protein-labeled cells inside an embryonic or postnatal heart can be imaged clearly and accurately.

Single clonal tracing and analysis at the whole-heart level can determine cardiac progenitor cell behavior and differentiation during cardiac development, ...

Analysis of Coronary Vessels in Cleared Embryonic Hearts

Whole mount visualization of the embryonic coronary plexus from which the capillary and arterial networks will form is rendered problematic using standard microscopy techniques, due to the scattering of imaging light by the thick heart tissue, as these vessels are localized deep within the walls of the developing heart. As optical clearing of tissues using organic solvents such as BABB (1 part benzyl alcohol to 2 parts benzyl benzoate) has been shown to greatly improve the optical penetration depth that can be achieved, we combined clearance of whole, PECAM1-immunostained hearts, with laser-scanning confocal microscopy, in order to obtain high-resolution images of vessels throughout the entire heart. BABB clearance of embryonic hearts takes place rapidly and also acts to preserve the fluorescent signal for several weeks; in addition, samples can be imaged multiple times without loss of signal. This straightforward method is also applicable to imaging other types of blood vessels in whole embryos.

We present a protocol for the analysis of coronary vessels in whole embryonic murine hearts up to E15.5, using standard immunological staining methods followed by optical clearance and confocal microscopy. This technique enables visualization of blood vessels throughout the entire heart without the need for time-consuming analysis of serial sections.

Whole mount visualization of the embryonic coronary plexus from which the capillary and arterial networks will form is rendered problematic using standard ...

Visualization of Cell Cycle Variations and Determination of Nucleation in Postnatal Cardiomyocytes

Cardiomyocytes are prone to variations of the cell cycle, such as endoreduplication (continuing rounds of DNA synthesis without karyokinesis and cytokinesis) and acytokinetic mitosis (karyokinesis but no cytokinesis). Such atypical cell cycle variations result in polyploid and multinucleated cells rather than in cell division. Therefore, to determine cardiac turnover and regeneration, it is of crucial importance to correctly identify cardiomyocyte nuclei, the number of nuclei per cell, and their cell cycle status. This is especially true for the use of nuclear markers for identifying cell cycle activity, such as thymidine analogues Ki-67, PCNA, or pHH3. Here, we present methods for recognizing cardiomyocytes and their nuclearity and for determining their cell cycle activity. We use two published transgenic systems: the Myh6-H2B-mCh transgenic mouse line, for the unequivocal identification of cardiomyocyte nuclei, and the CAG-eGFP-anillin mouse line, for distinguishing cell division from cell cycle variations. Combined together, these two systems ease the study of cardiac regeneration and plasticity.

To distinguish cell division from cell cycle variations in cardiomyocytes, we present protocols using two transgenic mouse lines: Myh6-H2B-mCh transgenic mice, for the unequivocal identification of cardiomyocyte nuclei, and CAG-eGFP-anillin mice, for distinguishing cell division from cell cycle variations.

Cardiomyocytes are prone to variations of the cell cycle, such as endoreduplication (continuing rounds of DNA synthesis without karyokinesis and ...

Assessing Cardiomyocyte Subtypes Following Transcription Factor-mediated Reprogramming of Mouse Embryonic Fibroblasts

Direct reprogramming of one cell type into another has recently emerged as a powerful paradigm for regenerative medicine, disease modeling, and lineage specification. In particular, the conversion of fibroblasts into induced cardiomyocyte-like myocytes (iCLMs) by Gata4, Hand2, Mef2c, and Tbx5 (GHMT) represents an important avenue for generating de novo cardiac myocytes in vitro and in vivo. Recent evidence suggests that GHMT generates a greater diversity of cardiac subtypes than previously appreciated, thus underscoring the need for a systematic approach to conducting additional studies. Before direct reprogramming can be used as a therapeutic strategy, however, the mechanistic underpinnings of lineage conversion must be understood in detail to generate specific cardiac subtypes. Here we present a detailed protocol for generating iCLMs by GHMT-mediated reprogramming of mouse embryonic fibroblasts (MEFs). 
We outline methods for MEF isolation, retroviral production, and MEF infection to accomplish efficient reprogramming. To determine the subtype identity of reprogrammed cells, we detail a step-by-step approach for performing immunocytochemistry on iCLMs using a defined set of compatible antibodies. Methods for confocal microscopy, identification, and quantification of iCLMs and individual atrial (iAM), ventricular (iVM), and pacemaker (iPM) subtypes are also presented. Finally, we discuss representative results of prototypical direct reprogramming experiments and highlight important technical aspects of our protocol to ensure efficient lineage conversion. Taken together, our optimized protocol should provide a stepwise approach for investigators to conduct meaningful cardiac reprogramming experiments that require identification of individual CM subtypes.

This manuscript describes a step-by-step protocol for the generation and quantification of diverse reprogrammed cardiac subtypes using a retrovirus-mediated delivery of Gata4, Hand2, Mef2c, and Tbx5.

Direct reprogramming of one cell type into another has recently emerged as a powerful paradigm for regenerative medicine, disease modeling, and lineage ...

Quantitative Whole-mount Immunofluorescence Analysis of Cardiac Progenitor Populations in Mouse Embryos

The use of ever-advancing imaging techniques has contributed broadly to our increased understanding of embryonic development. Pre-implantation development and organogenesis are two areas of research that have benefitted greatly from these advances, due to the high quality of data that can be obtained directly from imaging pre-implantation embryos or ex vivo organs. While pre-implantation embryos have yielded data with especially high spatial resolution, later stages have been less amenable to three-dimensional reconstruction. Obtaining high-quality 3D or volumetric data for known embryonic structures in combination with fate mapping or genetic lineage tracing will allow for a more comprehensive analysis of the morphogenetic events taking place during embryogenesis.
This protocol describes a whole-mount immunofluorescence approach that allows for the labeling, visualization, and quantification of progenitor cell populations within the developing cardiac crescent, a key structure formed during heart development. The approach is designed in such a way that both cell- and tissue-level information can be obtained. Using confocal microscopy and image processing, this protocol allows for three-dimensional spatial reconstruction of the cardiac crescent, thereby providing the ability to analyze the localization and organization of specific progenitor populations during this critical phase of heart development. Importantly, the use of reference antibodies allows for successive masking of the cardiac crescent and subsequent quantitative measurements of areas within the crescent. This protocol will not only enable a detailed examination of early heart development, but with adaptations should be applicable to most organ systems in the gastrula to early somite stage mouse embryo.

Here, we describe a protocol for whole mount immunofluorescence and image-based quantitative volumetric analysis of early stage mouse embryos. We present this technique as a powerful approach to qualitatively and quantitatively assess cardiac structures during development, and propose that it may be widely adaptable to other organ systems.

The use of ever-advancing imaging techniques has contributed broadly to our increased understanding of embryonic development. Pre-implantation development ...

Microinjection-based System for In Vivo Implantation of Embryonic Cardiomyocytes in the Avian Embryo

Interpreting the relative impact of cell autonomous patterning versus extrinsic microenvironmental influence on cell lineage determination represents a general challenge in developmental biology research. In the embryonic heart, this can be particularly difficult as regional differences in the expression of transcriptional regulators, paracrine/juxtacrine signaling cues, and hemodynamic force are all known to influence cardiomyocyte maturation. A simplified method to alter a developing cardiomyocyte's molecular and biomechanical microenvironment would, therefore, serve as a powerful technique to examine how local conditions influence cell fate and function. To address this, we have optimized a method to physically transplant juvenile cardiomyocytes into ectopic locations in the heart or the surrounding embryonic tissue. This allows us to examine how microenvironmental conditions influence cardiomyocyte fate transitions at single cell resolution within the intact embryo. Here, we describe a protocol in which embryonic myocytes can be isolated from a variety of cardiac sub-domains, dissociated, fluorescently labeled, and microinjected into host embryos with high precision. Cells can then be directly analyzed in situ using a variety of imaging and histological techniques. This protocol is a powerful alternative to traditional grafting experiments that can be prohibitively difficult in a moving tissue such as the heart. The general outline of this method can also be adapted to a variety of donor tissues and host environments, and its ease of use, low cost, and speed make it a potentially useful application for a variety of developmental studies.

In this method, embryonic cardiac tissues are surgically microdissected, dissociated, fluorescently labeled, and implanted into host embryonic tissues. This provides a platform for studying the individual or tissue level developmental organization under ectopic hemodynamic conditions, and/or altered paracrine/juxtacrine environments.

Interpreting the relative impact of cell autonomous patterning versus extrinsic microenvironmental influence on cell lineage determination represents a ...

Analysis of Cardiac Chamber Development During Mouse Embryogenesis Using Whole Mount Epifluorescence

The goal of this protocol is to describe a method for the dissection of mouse embryos and visualization of embryonic mouse ventricular chambers during heart development using ventricular specific fluorescent reporter knock-in mice (MLC-2v-tdTomato mice). Heart development involves a linear heart tube formation, the heart tube looping, and four chamber septation. These complex processes are highly conserved in all vertebrates. The mouse embryonic heart has been widely used for heart developmental studies. However, due to their extremely small size, dissecting mouse embryonic hearts is technically challenging. In addition, visualization of cardiac chamber formation often needs in situ hybridization, beta-galactosidase staining using LacZ reporter mice, or immunostaining of sectioned embryonic hearts. Here, we describe how to dissect mouse embryonic hearts and directly visualize ventricular chamber formation of MLC-2v-tdTomato mice using whole mount epifluorescent microscopy. With this method, it is possible to directly examine heart tube formation and looping, and four chamber formation without further experimental manipulation of mouse embryos. Although the MLC-2v-tdTomato reporter knock-in mouse line is used in this protocol as an example, this protocol can be applied to other heart-specific fluorescent reporter transgenic mouse lines.

We present the protocols to examine mouse heart development using whole mount epifluorescent microscopy on mouse embryos dissected from ventricular specific MLC-2v-tdTomato reporter knock-in mice. This method allows us to directly visualize each stage of the ventricular formation during mouse heart development without labor-intensive histochemical methods.

The goal of this protocol is to describe a method for the dissection of mouse embryos and visualization of embryonic mouse ventricular chambers during ...

Live Imaging of Early Cardiac Progenitors in the Mouse Embryo

The first steps of heart development imply drastic changes in cell behavior and differentiation. While analysis of fixed embryos allows studying in detail specific developmental stages in a still snapshot, live imaging captures dynamic morphogenetic events, such as cell migration, shape changes, and differentiation, by imaging the embryo as it develops. This complements fixed analysis and expands the understanding of how organs develop during embryogenesis. Despite its advantages, live imaging is rarely used in mouse models because of its technical challenges. Early mouse embryos are sensitive when cultured ex vivo and require efficient handling. To facilitate a broader use of live imaging in mouse developmental research, this paper presents a detailed protocol for two-photon live microscopy that allows long-term acquisition in mouse embryos. In addition to the protocol, tips are provided on embryo handling and culture optimization. This will help understand key events in early mouse organogenesis, enhancing the understanding of cardiovascular progenitor biology.

We present a detailed protocol for mouse embryo culture and imaging that enables 3D + time imaging of cardiac progenitor cells. This video-toolkit addresses the key skills required for successful live imaging otherwise hard to acquire from text-only publications.

The first steps of heart development imply drastic changes in cell behavior and differentiation. While analysis of fixed embryos allows studying in detail ...

Immunostaining of Dissected Zebrafish Embryonic Heart 

Zebrafish embryo becomes a popular in vivo vertebrate model for studying cardiac development and human heart diseases due to its advantageous embryology and genetics 1,2. About 100-200 embryos are readily available every week from a single pair of adult fish. The transparent embryos that develop ex utero make them ideal for assessing cardiac defects 3. The expression of any gene can be manipulated via morpholino technology or RNA injection 4. Moreover, forward genetic screens have already generated a list of mutants that affect different perspectives of cardiogenesis 5. 
Whole mount immunostaining is an important technique in this animal model to reveal the expression pattern of the targeted protein to a particular tissue 6. However, high resolution images that can reveal cellular or subcellular structures have been difficult, mainly due to the physical location of the heart and the poor penetration of the antibodies. 
 Here, we present a method to address these bottlenecks by dissecting heart first and then conducting the staining process on the surface of a microscope slide. To prevent the loss of small heart samples and to facilitate solution handling, we restricted the heart samples within a circle on the surface of the microscope slides drawn by an immEdge pen. After the staining, the fluorescence signals can be directly observed by a compound microscope. 
Our new method significantly improves the penetration for antibodies, since a heart from an embryonic fish only consists of few cell layers. High quality images from intact hearts can be obtained within a much reduced procession time for zebrafish embryos aged from day 2 to day 6. Our method can be potentially extended to stain other organs dissected from either zebrafish or other small animals.

Immunostaining of Dissected Zebrafish Embryonic Heart

A rapid way to conduct immunostaining of zebrafish embryonic heart is described. Compared to the whole mount immunostaining approach, this method dramatically increases the penetration of the antibodies, which allows obtaining high resolution images that reveal cellular/subcellular structures in the heart within a much reduced processing time.

Zebrafish embryo becomes a popular in vivo vertebrate model for studying cardiac development and human heart diseases due to its advantageous embryology ...

Biology

Isolation and Culture of Neonatal Mouse Cardiomyocytes

Cultured neonatal cardiomyocytes have long been used to study myofibrillogenesis and myofibrillar functions. Cultured cardiomyocytes allow for easy investigation and manipulation of biochemical pathways, and their effect on the biomechanical properties of spontaneously beating cardiomyocytes.	
The following 2-day protocol describes the isolation and culture of neonatal mouse cardiomyocytes. We show how to easily dissect hearts from neonates, dissociate the cardiac tissue and enrich cardiomyocytes from the cardiac cell-population. We discuss the usage of different enzyme mixes for cell-dissociation, and their effects on cell-viability. The isolated cardiomyocytes can be subsequently used for a variety of morphological, electrophysiological, biochemical, cell-biological or biomechanical assays. We optimized the protocol for robustness and reproducibility, by using only commercially available solutions and enzyme mixes that show little lot-to-lot variability. We also address common problems associated with the isolation and culture of cardiomyocytes, and offer a variety of options for the optimization of isolation and culture conditions.

Primary mouse cardiomyocyte cultures are one of the pivotal tools for the investigation of myofibrillar organization and function. The following protocol describes the isolation and culture of primary cardiomyocytes from neonatal mouse hearts. The resulting cardiomyocyte cultures may be subsequently used for a variety of biomechanical, biochemical and cell-biological assays.

Cultured neonatal cardiomyocytes have long been used to study  myofibrillogenesis and myofibrillar functions. Cultured cardiomyocytes allow  for easy ...

Analysis of Cardiomyocyte Development using Immunofluorescence in Embryonic Mouse Heart

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