Rabbit specific hrp aec ihc detection kit micro polymer

Primary renal, spleen, and renal allograft tissue cross-sections were fixed in 10% formalin and embedded in paraffin. Five μm sections were cut with a Leica CM1900 cryomicrotome (Leica Biosystems Division of Leica Microsystems Inc.). The sections underwent H&E, PAS, and Masson's staining for histological evaluation. Immunochemistry staining was performed following the manufacturer’s protocol. Briefly, sections were deparaffinized by successively washing them with xylene, 100% and 95% ethanol, and distilled water. After antigen unmasking and blocking, the sections were incubated with anti-C4d antibody (HP8033; Hycult Biotech, USA) or anti-IL21R antibody (ab5980; Abcam, UK) overnight at 4°C. The rabbit-specific HRP/AEC IHC Detection Kit Micro-polymer (Abcam, UK) was used to visualize the stained sections, followed by staining with diaminobenzidine and counterstaining with hematoxylin. The acute humoral rejection was confirmed by the presence of DSA, linear C4d staining in peritubular capillaries, and histologic evidence of graft injury [44 (link)]. A well-experienced pathologist scored the histopathologic characteristics of rejection, including tubulitis, glomerulitis, interstitial inflammation, intimal arteritis, and peritubular capillaritis according to the Banff lesion scores [45 (link)]. ImageJ software was used to analyze the captured images.

The skin at the vaccination site and tumors were harvested 3 and 6 days following the start of the treatment. Samples were left overnight in a formalin-free zinc fixative (BD Biosciences, San Jose, CA, USA) and then transferred into 70% ethanol. Skin and tumor samples were embedded in paraffin and 5 or 2 µm thick sections were cut, respectively. Samples were stained with hematoxylin and eosin (H&E) and observed at 40 × magnification using a BX-51 microscope (Olympus, Düsseldorf, Germany).
Immunohistochemical (IHC) staining was performed using the EXPOSE Rabbit HRP/AEC kit (ab64261; Abcam, Cambridge, UK) and Rabbit Specific HRP/AEC IHC Detection Kit-Micro-polymer (ab236468; Abcam, Cambridge, UK) following the manufacturer protocol. The primary Anti-Granzyme B antibody (ab4059; Abcam, Cambridge, UK) at 1:1000 dilution and the primary Foxp3 Antibody (5H10L18; Thermo Fisher Scientific, Waltham, MA, USA) at 1:1200 dilution were used. Samples were observed at 40 × objective magnification using the BX-51 microscope. For analysis, 10 images per sample were taken using a DP72 CCD camera connected to the microscope. The average number of Granzyme B positive (GrB+) and FoxP3 positive (FoxP3+) cells in a visual field was assessed in a blind fashion by three examiners [21 (link),36 (link)].