|
|
Blood, Vol. 104, Issue 10, 3228-3230, November 15, 2004
Visualization of lymphatic vessels through NF- B activity
Blood Saban et al.
104: 3228
Supplemental materials for: Saban et al, Vol 104, Issue 10, 3228-3230
Files in this Data Supplement:
- Figure S1. Illustration of morphometric analysis of lymphatic vessels.(JPG, 97 KB)
-
Tissues such as the bladder (A) were isolated from naive  B-lacZ mice, stained as whole mount with X-gal for 6 hours, photographed, and brought into Neurolucida software (MicroBrightField, Williston, VT) (A). A grid containing boxes of 15.625 microns was photographed at the same magnification of the whole mount (7.5X) and digitally added to the tissue section (A). This grid was used to calibrate the software and facilitated the tracing of lymphatic vessels. The length and diameter of vessels were digitally traced by calibrated pointer with adjustable diameter (B). The tracing software permits the placement of bifurcations and changed color as each order of bifurcation was selected. Therefore, the major caliber vessels were in red, the second order ones were traced in blue and third order vessels in orange. For the sake of clarity, in this particular illustration, only half of the vessels were traced, leaving the other half labeled with X-gal for comparison (B). Once all the vascular elements were traced, the software permitted the determination of tissue contours by tracing the entire region of interest (1B= violet line; blue arrow head). Results were analyzed with NeuroExplorer. Note that bladder lymphatic vessels seem to contribute to two major vessels of large caliber (black arrow head). Those main vessels were found in each site of the organ, running along the axis from the neck to dome. Original magnification, X 7.5. Note on alterations at the histologic level. A detailed morphometric analysis was performed by importing Neurolucida tracings into NeuroExplorer version 3.70.2 (MicroBrightField). The ratio between the area occupied by lymphatics and total tissue area (µm2) was then determined for each section and the statistical analysis was performed using Wilcoxon’s rank sum test. Results are expressed as mean ± SEM. In all cases, a P value of less than .05 was considered indicative of significant difference (Bernard R. Hypothesis testing for correlation coefficients. Boston: PWS Publishers; 1990). This method may result in overestimation of lymphatic area. The software uses the 3-dimensional surface area of lymphatics, estimated from a 2-dimensional projection. Also, tissue area was calculated from a 2-dimensional projected cross-sectional tissue area (tissue contour).
|
|
