(b) Anti-CD68 immunohistochemistry with USPIO particles counterstaining
shows several brown stained macrophages (white arrow heads) and blue iron particles within the structures (black arrows) which are consistent with positively stained vessels on anti-CD31 staining (magnification x200).
Panel (a) overlay of HBcAg (red) and EpCAM (green) positive cells and DAP1 nuclei counterstaining
(blue) in HCC tissue (panels (a1)-(a3), resp.).
was then performed with haematoxylin.
The reaction was visualized using diaminobenzidine followed by counterstaining
The denaturation and hybridization of the probe and the target DNA with recommended parameters of time and temperature, final post-hybridization washes and counterstaining
with 4'6-diamidino 2-phenylindole hydrochloride (DAPI) were carried out as per manufacturer's protocol on the patient slide.
After washing, images were visualized using 3,3'-diaminobendizine (DAB), with hematoxylin counterstaining
for 1 min.
with hematoxylin-eosin was performed for regular histologic characterization of the tumor.
TUNEL-positive nuclei were counted in ten nonoverlapping fields per coverslip and then converted to percentage by comparing TUNEL-positive counts with the total cell nuclei as determined by DAPI (4',6'-diamino-2-phenylindole) counterstaining
with hematoxylin was performed for nuclear staining.
Immunoreactions were detected and visualized with the polymer-peroxidasemethod (EnVision+/HRP and 3,3-diaminobenzidine; Dako Cytomation, Denmark) followed by counterstaining
with Mayer hematoxylin.
All steps were performed on Discovery XT Automated Immunostainer using VMSI validated reagents, including deparaffinization, cell conditioning (antigen retrieval with a borate-EDTA buffer), primary antibody staining, detection and amplification using a biotinylated-streptavidin-HRP and DAB (Diaminobenzidine) system, and hematoxylin counterstaining
. Following staining on the instrument, slides were dehydrated through graded alcohols to xylene and coverslipped with mounting medium.
was done using Brilliant Green for 20 s.