TITLE: FAST DiI Injection Protocol from Neural Tracing in Porcine SAN 
AUTHORS: Donald  Hoover, Peter    Hanna, Zixi  Jack Cheng

[DESCRIPTION]
Protocol for injecting FAST DiI into the sinoatrial node of porcine test subjects for neural tracing purposes. 

[STEPS]
SECTION: Injection of FAST DiI
1. Sedated, anesthetized, and intubated pig (induction: ketamine 10 mg/kg IM/midazolam 1 mg/ kg IM, maintenance: isoflurane 1-2% inhalation).
SECTION: Injection of FAST DiI
2. Performed right unilateral thoracotomy by dividing the pectoral muscle.
SECTION: Injection of FAST DiI
3. Made a small incision in the pericardium and exposed the right atrial (RA)-superior vena cava junction.
SECTION: Injection of FAST DiI
4. 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. 
SECTION: Injection of FAST DiI
5. Placed chest tube and closed incision.
SECTION: Injection of FAST DiI
6. Aspirated chest tube immediately prior to its removal. 
SECTION: Tissue Dissection and Preparation
7. Harvested tissue in a terminal procedure at least 3 weeks after injection procedure. 
SECTION: Tissue Dissection and Preparation
8. Immersed tissue in cold 4% PFA for 24 hours.
SECTION: Tissue Dissection and Preparation
9. Washed tissue in PBS 4 times. 
SECTION: Tissue Dissection and Preparation
10. Transferred tissue to cold 0.01M PBS + 0.02% Na azide + 20% sucrose solution for shipment to East Tennessee State University.
SECTION: Tissue Sectioning Using the Cryostat
13. Removed tissues from -80°C freezer and mounted onto specimen plate using Tissue-Tek® O.C.T. Compound (Sakura Finetek USA, Cat. No. 4583). 
SECTION: Tissue Dissection and Preparation
11. Dissected tissue as needed to isolate specific regions.
SECTION: Tissue Dissection and Preparation
12. Froze tissues on dry ice and stored at -80°C until ready to be sectioned.
SECTION: Tissue Sectioning Using the Cryostat
14. Cut tissues into 30μm sections at -20 to -25°C using a Leica CM3050S cryostat (Leica Microsystems Inc., Bannockburn, IL, USA).
SECTION: Tissue Sectioning Using the Cryostat
15. 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.
SECTION: Tissue Sectioning Using the Cryostat
16. Stored sets of tissues in slide boxes wrapped in aluminum foil at -20°C until further processing.
SECTION: Immunohistochemistry
17. 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)
SECTION: Immunohistochemistry
18. 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).
SECTION: Imaging
19. 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).
SECTION: Imaging
20. Loaded slide onto stage and collected representative images of ganglia.
SECTION: Quantification
21. Counted DiI-labeled neurons and total number of neurons (i.e., PGP9.5-positive).