Application software

Cross sections were cut from the central part of the Loop and Lectin IHC was performed. The newly grown vessels in the granulation tissue in the AV loop were Lectin IHC positive, appeared in brown lining in lumens. After the pictures of each slide were taken by camera with the Leica application software, the area of cross section was measured by Leica application software. The absolute vessel number was counted manually and by dividing it to the cross section area, the vessel density was calculated as well.
The slides were cooked in pressure cooker for 1 min for antigen retrieval. Isolectin B4 Biotinylated antibody (Bandeiraea Simplicifolia Sigma L2140) was diluted to 1:250 with Tris buffer, incubated in 2 ~ 8°C fridge overnight, following the reaction with Streptavidin-peroxidase for 30 min. The signal was developed by DAB for approximately 10 min.

Preparation for immunohistochemical staining, including sections deparaffinization, and antigens retrieving was conducted. After blocking non-specific protein binding, sections were washed by PBS and incubated overnight (at 4 °C) with the primary rabbit antibody against rat; cleaved caspase-3 (NB100-56113) from Novus Biologicals, (dilution 1:1000), or Bcl-2 (ab194583) from Abcam Co (Cambridge, MA, USA), (dilution 1:100). Then, sections were washed by PBS, incubated with secondary antibody; HRP Envision kit (DAKO), for 20 min, washed by PBS, and positive immunoreactivities were developed by DAB visualization for 10 min and counter-staining with hematoxylin. Quantitative analysis was performed according to El-Nabarawy et. al. (2020) for determination of area percentage of immunohistochemical expression levels of indicated proteins, as estimated from six representative randomly selected fields in the tissue section using Leica application software (Leica Microsystems GmbH, Wetzlar, Germany) [33 (link)]. Statistical analysis of renal immuno-expressions in different groups was carried out using chi-squared "χ²" test. Representative microscopic images (× 400) were shown in the study.