Analysis of Cardiomyocyte Development using Immunofluorescence in Embryonic Mouse Heart

The overall goal of this procedure is to assess myo fial and cardiomyocyte maturation in the developing embryonic mouse heart. This is accomplished by first orienting and freezing the embryonic heart in cutting medium. Next, the heart is cryo sectioned in the proper orientation.

Then the heart sections undergo immunofluorescent labeling of proteins of interest. Finally, confocal microscopy of immuno stain heart sections is carried out. Ultimately, two dimensional and three dimensional image analyses are used to show the development of MyFi and other cardiomyocyte structures.

This method can help answer key questions in the cardiac development field, such as how mutations and cardiac genes affect MyFi assembly and the emergence of specific structures such as interated discs and costumers. To snap freeze embryonic hearts use optimal cutting temperature or OCT medium to fill a 3.5 centimeter Petri dish in a chemical hood.Cool. Two methyl butane in liquid nitrogen.

After isolating mouse embryonic hearts according to the text protocol, place the hearts in OCT and allow them to equilibrate for several seconds before transferring them to a seven millimeter mold containing OCT. Orient the interior wall of the heart to the bottom of the mold. Gently place the mold into the liquid nitrogen cooled two methyl butane.

Taking care not to allow the two methyl butane 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. After all the hearts are frozen, wrap the cryo molds in foil and store it negative 80 degrees Celsius until ready for cryosectioning.

To fix embryonic hearts, use 4%PFA in PBS to fill the wells of a 12 foil culture plate. After dissecting out the embryonic hearts according to the text protocol, place each heart into a well of PFA and fix at four degrees Celsius overnight To cryo protect the hearts using a plastic transfer pipette, move each heart to a 1.5 milliliter micro centrifuge tube containing 1.5 milliliters of 15%Sucrose in PBS and gently agitate at four degrees Celsius until the heart sinks to the bottom of the tube. Transfer each heart to 30%sucrose in PBS and gently agitate at four degrees Celsius until the heart sinks to the bottom of the tube.

Freeze the embryos in OCT as demonstrated earlier in this video. After placing cryo molds into the cryostat chamber and equating to negative 17 degrees Celsius, invert the cryo mold 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.

Place a large drop of OCT onto the chuck and mount the heart block onto the OCT drop. Keeping the orientation such that the anterior wall of the heart is furthest from the chuck. Allow the heart to freeze onto the chuck.

Load the chuck and mounted heart block onto the cryostat object holder. Adjust so that the angle of the blade is three to five degrees relative to the sample. Collect 10 micrometer sections onto microscope slides that have been pretreated with a positively charged coating.

Allow the samples to dry completely before storing at negative 80 degrees Celsius to carry out immunofluorescence. After fixing and or removing OCT from sections, use one x blocking buffer diluted in PBS to block for 45 minutes with gentle shaking. If using a primary antibody generated in mouse, add a donkey or goat anti mouse IgG H plus L monovalent FAB fragment diluted one to 100 in PBS 0.1%tween 20 and incubate at room temperature for 45 minutes with gentle shaking.

Add primary antibody or antibodies diluted in one x blocking buffer, and incubate for two hours at room temperature or at four degrees Celsius overnight. After the incubation, use one XPBS to wash the sections three times for 10 minutes. At room temperature addor conjugated secondary antibody diluted one to 500 in blocking buffer to the samples and incubate protected from light for two hours.

At room temperature, use one XPBS to wash the sections at room temperature three times for 10 minutes protected from light. Mount the slides at anti fade medium by placing two drops of medium on each end of the slide and use a cover slip to cover. Use nail polish to seal the cover slips.

Store it four degrees Celsius, protected from light until ready to image. Using a four x objective and laser fluorescence, find the sample and area of interest. Capture the image to use as a map.

When imaging at high magnification. Remove the slide making minimal adjustments to the slide stage. Then change to a 60 x oil immersion objective.

Place a small drop of oil on the objective and replace the slide onto the slide stage. Next, find the sample again, set the laser power exposure time and binning to the desired levels for each channel. Once optimal settings are determined according to the guidelines of the text protocol, use the sample settings for all tissue sections within the experiment.

Use the intensity histogram to note the optimal intensity range for each channel, which will be used for analysis. Generate a Zack using the acquisition function by selecting the appropriate laser channels. Then choose the upper and lower limits of the Z stack.

Choose a Zack step size that is one half the value of the optical slice thickness provided by the software. Click run to collect the images using Fiji or a comparable program for image analysis. Open the Zack file with a custom color mode option and the channels split into separate windows.

From the image pulldown menu, open the adjust brightness contrast tool and within each channel set the optimal histogram intensity range. As previously determined, apply these channel ranges to all Zacks being analyzed. Then from the image color, pull down menu, merge the individual channels into a single composite image.

Using the image Stacks Z project menu create a flattened Z stack from the composite image. Since 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 Zack To generate a 3D image, first choose the image stacks 3D project menu. 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 one. Then open the image J 3D viewer from the plugins pull down menu.

Choose the composite image generated display as volume and set the resampling factor to one or two. These figures show typical results for cos staining of different proteins in a snap frozen, an acetone fixed heart, the antibody against S alpha actinin reproducibly labeled Z discs, and in interated discs with high specificity and minimal background. The antibody against adherence junction protein beta-catenin bound the membrane of both cardiomyocytes and non cardiomyocyte cells and colocalization with SFA actinin occurred in presumed interated discs at E 16.5 beta one Integrin immunofluorescence in the embryonic heart is especially challenging, but beta one integrin staining in these studies revealed signal with the same periodicity as SFA Actinin labeled Z discs, possibly reflecting nascent costumers forming at E 16.5.

At E 12.5, the SFA actinin and tropo mycin staining pattern showed regular periodicity in trabecular cardiomyocytes consistent with mature myofibrils in the outer compact zone SFA actinin was more punctate than linear and the tropo myosin signal was diffuse rather than linear NCA adherent staining and trabecular cardiomyocytes at E 12.5 hearts co localized with areas of intense SFA actinin staining possibly representing interated discs. Shown here is A PFA fixed E 12.5 embryonic heart labeled for SFA actinin and filamentous acton from a life act RFP Ruby transgenic mouse. The SFA actin signaled to noise ratio was decreased compared to snap frozen heart sections.

Three dimensional image reconstruction revealed that MyFi within a cardiomyocyte were roughly parallel to one another, but individual cardiomyocytes were oriented at varying angles relative to one another. After watching this video, you should have a good understanding of how to properly orient and cryo section embryonic hearts, as well as perform immuno staining, confocal microscopy, and image analysis to assess MyFi and cardiomyocyte maturation during mouse heart development.Admit.