Biotin free polymer detection system

Ultrasound-guided needle allograft biopsies were performed as previously described [16 (link)]. Occasionally, biopsies were performed during an open re-operation under direct visualization. Protocol biopsies were performed at 1, 3, 6, and 12 months following ABO- and HLA-incompatible live donor transplants, but not routinely after deceased donor transplants or compatible live donor transplants. C4d staining was performed on frozen tissue using indirect immunofluorescence with anti-human C4d antibody (Quidel, San Diego, CA) at 1:40 dilution followed by secondary antibody (fluorescein isothiocyanate–conjugated goat anti-mouse IgG; Jackson Immunoresearch Laboratories, West Grove, PA) or on paraffin-embedded tissue sections using a rabbit polyclonal anti-human antibody (American, Pfungstadt, Germany) at a 1:50 dilution, in concert with a biotin-free polymer detection system (Leica, Wetzlar, Germany).

Ultrasound-guided needle allograft biopsies were performed as previously described (10 (link)), with biopsies occasionally performed under direct visualization during an open re-operation. At our institution, we perform protocol biopsies at 1, 3, 6, and 12 months following ABO-incompatible and/or HLA-incompatible live donor transplants, but do not routinely perform them for other transplant types.
Staining for C4d was performed on frozen tissue by indirect immunofluorescence using anti-human C4d antibody (Quidel, San Diego, CA) at a 1:40 dilution, followed by secondary antibody (fluorescein-isothiocyanate-conjugated goat antimouse IgG, Sigma-Aldrich) or on paraffin embedded tissue sections using a rabbit polyclonal anti-human antibody at a 1:50 dilution and a biotin-free polymer detection system (Lecia).