Jun 01, 2020
Sectioning of Mouse Proximal Thoracic Aorta
- Jeff Z. Chen1,
- Deborah A. Howatt1,
- Jessica J. Moorleghen1,
- Michael K. Franklin1,
- Hisashi Sawada1,
- Yanxiang Gao1,
- Hong S. Lu1,
- Alan Daugherty1
- 1University of Kentucky
External link: http://cvrc.med.uky.edu/lab-protocols
Protocol Citation: Jeff Z. Chen, Deborah A. Howatt, Jessica J. Moorleghen, Michael K. Franklin, Hisashi Sawada, Yanxiang Gao, Hong S. Lu, Alan Daugherty 2020. Sectioning of Mouse Proximal Thoracic Aorta. protocols.io https://dx.doi.org/10.17504/protocols.io.be9mjh46
Manuscript citation:
Chen JZ, Sawada H, Moorleghen JJ, Weiland M, Daugherty A, Sheppard MB. Aortic Strain Correlates with Elastin Fragmentation in Fibrillin-1 Hypomorphic Mice. Circ Rep. 2019;1(5):199–205. doi:10.1253/circrep.CR-18-0012
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: April 17, 2020
Last Modified: June 01, 2020
Protocol Integer ID: 35853
Keywords: Aorta, Histology, Cryosection, Immunohistochemistry, Immunofloresance , Aneurysm,
Abstract
This protocol is used for generation of ascending aortic serial sections for histology.
This protocol generates a set of 10 slides covering ~1mm of ascending aortic tissue.
Inclusion of the descening aorta provides orientation and a non-TAA control from the same animal.
Attachments
Materials
1. Dissecting scissors, forceps, scalpel blade, and Parafilm
2. Tissue molds 15x15 mm (Fisher Scientific Cat # 41-741) and microcentrifuge tube with screw top.
3. OCT compound (Fisher Scientific Cat # 14-373-65)
4. Probe-on-Plus microscope slides (Fisher Scientific Cat # 22-230-900)
Safety warnings
Be careful when using the cryostat, blade is sharp.
Before start
1. OCT will become rubbery and difficult to cut if frozen for long periods. If you plan to cut the tissues within a few weeks, place aortic root tissues in tissue molds covered with OCT compound, wrapped tightly in parafilm, and store at -20̊C. Otherwise, store aortic roots covered by OCT compound in microcentrifuge tube with screw top at -20 ̊C for long-term storage. Label tissue molds or microcentrifuge tubes with the study name, date, and mouse ID or code as appropriate.
2. After anesthesia, cut open the thoracic cavity and draw blood from the right ventricle. Cut open the right atrium. Perfuse with saline from the apical side of the left ventricle to remove blood from the systemic circulation. A good sign for successful removal of blood from the systemic circulation is visualization of the liver turning from red to pale.
Collection of thoracic aorta
Collection of thoracic aorta
15m
15m
Euthanize mouse with ketamine : xyalazine cocktail
15m
Open thoracic cavity and expose heart
Nick right atrium with sharp scissors
Flush vasculature with cold saline (>5 mL) introduced slowly into the left ventricle
Dissect away the thymus, lungs, trachea, and esophagus
Inject optimal cutting temperature compound (OCT; ~0.5 mL) slowly into the left ventricle to keep patent, inject until the lumen of the descending aorta expands
Dissect the heart and thoracic aorta by ligating the left/right subclavians and common carotids near the thoracic outlet and ligating the aorta above the diaphragm
Dissect the descending aorta from the spine, taking care not to nick the aorta
Preparing tissue block
Preparing tissue block
Put a thin layer of OCT in mold
Preparing tissue block
Preparing tissue block
Separate the heart from the aorta by holding the heart with the forceps and cutting with either scissors or a scalpel blade, as close to the heart as possible without causing damage.
Figure 1: Approximate line to cut ventricle
Preparing tissue block
Preparing tissue block
Cut away approximately 60 - 70% of the ventricles (the red line shown in the Fig 1)
Preparing tissue block
Preparing tissue block
Mount the heart, ascending aorta, and descending aorta with the heart facing the left and descending aorta on the right. With permanent marker, mark where the aorta lays in the mold
Cover tissue with OCT. Place in cryostat with peltier on for 00:15:00 for OCT to solidify.
15m
Preparing the tissue block and cryostat for cutting
Preparing the tissue block and cryostat for cutting
Set temperature to -20 °C
Set chuck advance to 10 µm
Make sure stage is level
Make sure sectioning starts with new region of blade or new blade
Make sure anti roll plate is not chipped
Label 10 slides / sample / region. For example: Study name, mouse ID and group, region collected (ascending or root) and number of slide (1-10)
Remove block from mold
Trim excess solid OCT from tissue, keeping intact the descending aorta
Cut the heart at the mid ventricle level
Put small drop of OCT onto mount
Mount trimmed tissue block with the aortic arch facing up onto holder
Place in peltier and wait 10 minutes for interface to solidify
Cutting Sections of the Aorta
Cutting Sections of the Aorta
Note
The method requires cutting sequential sections in the correct orientation and without tearing, curling, or sticking incorrectly to the slide. Therefore, do not cut aortas collected from your experiments until you are sure you can reliably cut sections sequentially
Place holder onto cryostat chuck with the heart facing down and the descending aorta on top.
Start sectioning, checking every 10 cycles until tissue can be seen.
Start collecting serial sections when arch divides into ascending aorta and descending aorta. Ascending aorta should be towards you and descending aorta should be away.
Note
This should produce sections with both the ascending aorta and descending aorta. Ascending aortas should be towards the top of the slide, descending aortas should be towards the bottom. Verify orientation of sections by counting the number of elastic laminae seen: Ascending = 8-10 lamina, Descending = 8-6 lamina.
Collect at least 9 serial sections per slide. (Fig 2)
Figure 2: The organization of sections on consecutive slides
Note
Collect first section produced on bottom left corner of slide 1. Collect second section on bottom left corner of slide 2 and subsequent section in this mode. Collect eleventh section to the right of first section on slide 1. Collect serial sections by holding labeled slide downward with the label facing you. The ascending aorta should be on towards the top of the slide and the descending on bottom of slide
If section rolls or flips - use antistatic brush to unroll section
Stop collecting at the level of the aortic root - where cusps of valve leaflets are seen. Serial sections are collected at 10 μm/section, and 8 to 10 slides/aorta until the aortic wall disappears or is not intact anymore. Usually at least 9 serial sections/slide can be collected.
Clean up cryostat
Clean up cryostat
Lock chuck wheel
Remove block from chuck and mount - save or discard excess tissue
Brush clean all surfaces with 70% ethanol and brush
Discard trimmings
Turn off light
Close cryostat window
Storage
Storage
Slides should be stored in a appropriately labeled slide box (both the top and one of the short edges of the box) at -20̊C.