Aug 02, 2022
FAST DiI Injection Protocol from Neural Tracing in Porcine SAN
- Donald Hoover1,
- Peter Hanna2,
- Zixi (Jack) Cheng2
- 1East Tennessee State University;
- 2University of California, Los Angeles
Protocol Citation: Donald Hoover, Peter Hanna, Zixi (Jack) Cheng 2022. FAST DiI Injection Protocol from Neural Tracing in Porcine SAN . protocols.io https://dx.doi.org/10.17504/protocols.io.4r3l2okqqv1y/v1
Manuscript citation:
Hanna P, Dacey MJ, Brennan J, Moss A, Robbins S, Achanta S, Biscola NP, Swid MA, Rajendran PS, Mori S, Hadaya JE, Smith EH, Peirce SG, Chen J, Havton LA, Cheng ZJ, Vadigepalli R, Schwaber J, Lux RL, Efimov I, Tompkins JD, Hoover DB, Ardell JL, Shivkumar K. Innervation and Neuronal Control of the Mammalian Sinoatrial Node a Comprehensive Atlas. Circ Res. 2021 Apr 30;128(9):1279-1296. doi: 10.1161/CIRCRESAHA.120.318458. Epub 2021 Feb 25. PMID: 33629877; PMCID: PMC8284939.
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: July 18, 2022
Last Modified: August 02, 2022
Protocol Integer ID: 66991
Funders Acknowledgement:
Kalyanam Shivkumar
Grant ID: OT2 OD023848
Abstract
Protocol for injecting FAST DiI into the sinoatrial node of porcine test subjects for neural tracing purposes.
Materials
FAST DiI from Invitrogen, catalog D7756
CitiFluor AF1 Mounting Medium Solution (Electron Microscopy Sciences, Hatfield, PA, USA, Cat. #17970-100
Injection of FAST DiI
Injection of FAST DiI
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.
Tissue Dissection and Preparation
Tissue Dissection and Preparation
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.
Tissue Sectioning Using the Cryostat
Tissue Sectioning Using the Cryostat
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.
Immunohistochemistry
Immunohistochemistry
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).
Imaging
Imaging
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.
Quantification
Quantification
Counted DiI-labeled neurons and total number of neurons (i.e., PGP9.5-positive).