FAST DiI Injection Protocol from Neural Tracing in Porcine SAN

Sedated, anesthetized, and intubated pig (induction: ketamine 10 mg/kg IM/midazolam 1 mg/ kg IM, maintenance: isoflurane 1-2% inhalation).

Performed right unilateral thoracotomy by dividing the pectoral muscle.

Made a small incision in the pericardium and exposed the right atrial (RA)-superior vena cava junction.

Used 27-guage needle to inject 5 mg of an analog of 1,1'-dioctadecyl-3,3,3',3'- tetramethylindocarbocyanine perchlorate (FAST DiI, Invitrogen, D7756) in 250 μL of 100% methanol (MeOH) into the sinoatrial nodal (SAN) region.

Placed chest tube and closed incision.

Aspirated chest tube immediately prior to its removal.

Harvested tissue in a terminal procedure at least 3 weeks after injection procedure.

Immersed tissue in cold 4% PFA for 24 hours.

Washed tissue in PBS 4 times.

Transferred tissue to cold 0.01M PBS + 0.02% Na azide + 20% sucrose solution for shipment to East Tennessee State University.

Dissected tissue as needed to isolate specific regions.

Froze tissues on dry ice and stored at -80°C until ready to be sectioned.

Removed tissues from -80°C freezer and mounted onto specimen plate using Tissue-Tek® O.C.T. Compound (Sakura Finetek USA, Cat. No. 4583).

Cut tissues into 30μm sections at -20 to -25°C using a Leica CM3050S cryostat (Leica Microsystems Inc., Bannockburn, IL, USA).

Collected sections on charged slides in a sequence that yields at least 4 sets of tissue sections per region of the heart, with each set spanning the entire thickness of the specimen.

Stored sets of tissues in slide boxes wrapped in aluminum foil at -20°C until further processing.

Removed every second slide from a set to perform immunolabel for PGP9.5. (Described in separate protocol, found here: dx.doi.org/10.17504/protocols.io.8rmhv46)

Applied cover glass to adjacent tissue slides using a drop of CitiFluor AF1 Mounting Medium Solution (Electron Microscopy Sciences, Hatfield, PA, USA, Cat. #17970-100).

Imaged tissue sections using fluorescence illumination with an Olympus BX41 microscope equipped with an Olympus DP74 digital camera and cellSens software (Olympus America Inc., Center Valley, PA; RRID:SCR_016238).

Loaded slide onto stage and collected representative images of ganglia.

Counted DiI-labeled neurons and total number of neurons (i.e., PGP9.5-positive).