TITLE: Sectioning of Mouse Proximal Thoracic Aorta
AUTHORS: Jeff Z. Chen, Deborah A. Howatt, Jessica J. Moorleghen, Michael K. Franklin, Hisashi Sawada, Yanxiang Gao, Hong S. Lu, Alan Daugherty

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol is used for generation of ascending aortic serial sections for histology.</div><div class = "text-block">This protocol generates a set of 10 slides covering ~1mm of ascending aortic tissue.</div><div class = "text-block">Inclusion of the descening aorta provides orientation and a non-TAA control from the same animal.</div></div>

[STEPS]
?. [Collection of thoracic aorta]
Euthanize mouse with ketamine : xyalazine cocktail
?. [Collection of thoracic aorta]
Open thoracic cavity and expose heart
?. [Collection of thoracic aorta]
Nick right atrium with sharp scissors
?. [Collection of thoracic aorta]
Flush vasculature with cold saline (>5 mL) introduced slowly into the left ventricle
?. [Collection of thoracic aorta]
Dissect away the thymus, lungs, trachea, and esophagus
?. [Collection of thoracic aorta]
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
?. [Collection of thoracic aorta]
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
?. [Collection of thoracic aorta]
Dissect the descending aorta from the spine, taking care not to nick the aorta
?. [Preparing tissue block]
Put a thin layer of OCT in mold
?. [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.
?. Cut away approximately 60 - 70% of the ventricles.(the red line shown in the Figure)
?. [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
?. [Preparing tissue block]
Cover tissue with OCT. Place in cryostat with peltier on for for OCT to solidify.
?. [Preparing the tissue block and cryostat for cutting]
Set temperature to
-20 °C
?. [Preparing the tissue block and cryostat for cutting]
Set chuck advance to
?. [Preparing the tissue block and cryostat for cutting]
Make sure stage is level
?. [Preparing the tissue block and cryostat for cutting]
Make sure sectioning starts with new region of blade or new blade
?. [Preparing the tissue block and cryostat for cutting]
Make sure anti roll plate is not chipped
?. [Preparing the tissue block and cryostat for cutting]
Label 10 slides / sample / region. For example: Study name, mouse ID and group, region collected (ascending or root) and number of slide (1-10)
?. [Preparing the tissue block and cryostat for cutting]
Remove block from mold
?. [Preparing the tissue block and cryostat for cutting]
Trim excess solid OCT from tissue, keeping intact the descending aorta
?. [Preparing the tissue block and cryostat for cutting]
Cut the heart at the mid ventricle level
?. [Preparing the tissue block and cryostat for cutting]
Put small drop of OCT onto mount
?. [Preparing the tissue block and cryostat for cutting]
Mount trimmed tissue block with the aortic arch facing up onto holder
?. [Preparing the tissue block and cryostat for cutting]
Place in peltier and wait 10 minutes for interface to solidify
?. [Cutting Sections of the Aorta]
Place holder onto cryostat chuck with the heart facing down and the descending aorta on top.
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
?. [Cutting Sections of the Aorta]
Start sectioning, checking every 10 cycles until tissue can be seen.
?. [Cutting Sections of the Aorta]
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.
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.
?. [Cutting Sections of the Aorta]
Collect at least 9 serial sections per slide. (Fig 2)
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
?. [Cutting Sections of the Aorta]
If section rolls or flips - use antistatic brush to unroll section
?. [Cutting Sections of the Aorta]
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]
Lock chuck wheel
?. [Clean up cryostat]
Remove block from chuck and mount - save or discard excess tissue
?. [Clean up cryostat]
Brush clean all surfaces with 70% ethanol and brush
?. [Clean up cryostat]
Discard trimmings
?. [Clean up cryostat]
Turn off light
?. [Clean up cryostat]
Close cryostat window
?. [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.
?. [Preparing tissue block]
Cut away approximately 60 - 70% of the ventricles (the red line shown in the Fig 1)