The ablated veins harvested after autopsy were irrigated with normal saline. They were immersed and shaded in a 2% 2,3,5-triphenyltetrazolium chloride (TTC) (Sigma) solution and incubated for 1 hour at 40 ℃. After the staining was completed, the veins were sectioned longitudinally and completely unfolded. The exposed ablated site was macroscopically checked, and photographs were taken.
TTC-stained femoral/cephalic veins were fixed in 10% neutral-buffered formalin. Each fixed tissue was rinsed in tap water for 24 hours to completely remove the fixative from the tissue. For tissue dehydration, the tissue was gradually dehydrated using high-concentration ethanol of 70%–100%, and then a paraffin block was produced by clearing with xylene. The prepared block was cut to a thickness of 5 µm using a microtome to prepare slides. The slides were stained with H&E for microscopic evaluation.
Verifying the nonstained area in the vein subjected to TTC staining identified the surviving and damaged areas in the venous endothelium, making it easier to select the area to be examined under the microscope. The part that was not stained with TTC was assessed as the part where vein injury occurred through ablation.
The vessel injury score analyzed based on H&E staining was also used to objectively evaluate the ablating effect. Vessel injury scores were measured at 3 sites per harvested ablated vein. After scanning the entire tissue made of slides with a scanner, the damaged area was visually checked. This method was applied by modifying that of a previous study [3 (link)]. The criteria were assigned according to injury severity from 1 (least injury) to 4 (most injury): 1, endothelial cell coverage; 2, medial smooth muscle cell loss; 3, internal and external elastic lamina disruption; and 4, adventitia disruption. Scoring was comprehensively performed by a pathologist through evaluating the damaged area that each criterion had inflicted on the tissue.
TTC-stained femoral/cephalic veins were fixed in 10% neutral-buffered formalin. Each fixed tissue was rinsed in tap water for 24 hours to completely remove the fixative from the tissue. For tissue dehydration, the tissue was gradually dehydrated using high-concentration ethanol of 70%–100%, and then a paraffin block was produced by clearing with xylene. The prepared block was cut to a thickness of 5 µm using a microtome to prepare slides. The slides were stained with H&E for microscopic evaluation.
Verifying the nonstained area in the vein subjected to TTC staining identified the surviving and damaged areas in the venous endothelium, making it easier to select the area to be examined under the microscope. The part that was not stained with TTC was assessed as the part where vein injury occurred through ablation.
The vessel injury score analyzed based on H&E staining was also used to objectively evaluate the ablating effect. Vessel injury scores were measured at 3 sites per harvested ablated vein. After scanning the entire tissue made of slides with a scanner, the damaged area was visually checked. This method was applied by modifying that of a previous study [3 (link)]. The criteria were assigned according to injury severity from 1 (least injury) to 4 (most injury): 1, endothelial cell coverage; 2, medial smooth muscle cell loss; 3, internal and external elastic lamina disruption; and 4, adventitia disruption. Scoring was comprehensively performed by a pathologist through evaluating the damaged area that each criterion had inflicted on the tissue.
