Lectin angiography was performed in the AG (n = 5), GHNF (n = 5), and Silicone (n = 5) groups at 6 weeks after pretreatment31
. Mice were sedated, and 100 μg/body DyLight® 488 Lycopersican esculentum agglutinin (LEA, tomato lectin; Vector Labs, Burlingame, CA) was injected via the tail vein. Five minutes after lectin injection, animals were perfused through the heart using Na-PO4 buffered 4% paraformaldehyde. Following perfusion, tissues were washed with phosphate-buffered saline and transferred to 30% sucrose.
Tissues were cut using a microtome (thickness, 50 μm). Sections were examined under a confocal microscope (LSM780; Carl-Zeiss, Oberkochen, Germany). The same controls for brightness and exposure times were used among groups. Images were imported into Zeiss IMARIS (Carl-Zeiss, Germany) for volumetry. The vascular volume in subcutaneous capsules surrounding the silicone spacer or GHNF was calculated. To determine the density of blood vessels, the vascular volume was divided by the capsular volume. The brightness value of the vascular area was defined as >20, and the minimum volume was <5000 μm3 to extract artifacts.
. Mice were sedated, and 100 μg/body DyLight® 488 Lycopersican esculentum agglutinin (LEA, tomato lectin; Vector Labs, Burlingame, CA) was injected via the tail vein. Five minutes after lectin injection, animals were perfused through the heart using Na-PO4 buffered 4% paraformaldehyde. Following perfusion, tissues were washed with phosphate-buffered saline and transferred to 30% sucrose.
Tissues were cut using a microtome (thickness, 50 μm). Sections were examined under a confocal microscope (LSM780; Carl-Zeiss, Oberkochen, Germany). The same controls for brightness and exposure times were used among groups. Images were imported into Zeiss IMARIS (Carl-Zeiss, Germany) for volumetry. The vascular volume in subcutaneous capsules surrounding the silicone spacer or GHNF was calculated. To determine the density of blood vessels, the vascular volume was divided by the capsular volume. The brightness value of the vascular area was defined as >20, and the minimum volume was <5000 μm3 to extract artifacts.
