Whole-Mount Immunofluorescence Staining, Confocal Imaging and 3D Reconstruction of the Sinoatrial and Atrioventricular Node in the Mouse

This protocol explains the necessary steps to perform micro dissection whole-mount immunofluorescence staining and microscopy specifically for the sinus node and AV node in the mouse. The whole-mount methodology is advantageous for addressing the exact 3D localization and morphology of the sinus and AV node and to examine the relationship with the surrounding tissue. The tissue morphology is considerably preserved with only minimum tissue dehydration and physical rupture.

To begin, puncture the heart with a 27 gauge needle in the area of the apex by carefully pushing the needle into the left ventricle and gently injecting five to 10 milliliters of ice cold PBS to profuse the heart. Carefully lift the heart using a tweezer and cut the large vessels to remove the heart. Put the heart into a dissection dish filled with ice cold PBS under the dissecting microscope.

After determination of the orientation of the heart turn the heart around so that the front of the heart is at the bottom of the dish. Immobilize the heart by putting little pins through the apex and the atrial appendage into the agarose at the bottom of the dissection dish. Using fine tweezers and scissors carefully remove non-cardiac tissue around the superior and inferior vena cava.

To expose the intercaval region, remove the majority of the ventricles by cutting parallel to the groove between the ventricles and the atria with a micro scissors. For fixation and dehydration put the sample in 4%paraformaldehyde overnight at four degrees Celsius. On the next day, transfer the heart to 15%sucrose solution and incubate it for 24 hours at four degrees Celsius.

After the incubation transfer the heart to 30%sucrose solution for another 24 hours at four degrees Celsius. Wash the heart in 1%Triton X-100 diluted in PBS. Then block and permeabilize it in blocking solution overnight at four degrees Celsius.

Place the heart in a 1.5 milliliter tube and incubate it with rabbit anti-mouse Connexin 43 and rat anti-mouse HTN4 antibodies diluted with blocking solution for seven days at four degrees Celsius. After seven days, remove the solution containing primary antibodies using a pipette. Wash the heart with 1%Triton X-100 solution three times for one hour per wash on the orbital shaker.

Repeat the washing steps after the incubation with secondary antibodies and DAPI. After preparing plastic rings and filling them with plasticine, place the heart into the plasticine groove with the back of the heart facing up. Identify the SAN, which is located on the dorsal side of the heart with the intercaval region.

Add PBS to the heart to displace all air within the cavity until the heart is fully covered with PBS. Apply silicone to the edges of the plastic ring. Gently press the surrounding part of the sample into the plasticine to fix the heart and cover the heart with a cover slip.

Then gently press the backside of the plasticine to squeeze out some of the PBS and attach the heart to the cover slip taking care to avoid any air bubbles within the imaging area. Position the whole-mount staining samples upside down on the platform of the confocal microscope and proceed with imaging as described in the text manuscript. The parameters for each channel of the confocal microscope are set using the respective software.

This protocol was used to perform confocal microscopy imaging of both the sinoatrial node and atrioventricular node in murine hearts. Specific staining of the conduction system and working myocardium with fluorescent antibodies, targeting HCN4 and Connexin 43 respectively makes it possible to identify the sinoatrial node and the atrioventricular node within the intact anatomy. This whole-mount staining protocol makes it possible to study the interaction of different cell types by using various antibodies.