The tissue sections were deparaffinized and were then rehydrated and blocked with 3% H2O2 in methanol followed by antigen retrieval in a microwave in 10 mM citrate buffer, pH 6.0. Tissue sections were treated with universal blocking solution (BioGenex, HK085–5K) for 10 min, and then incubated overnight at 4°C with the following primary antibodies: anti-XBP1 (Santa Cruz Biotechnology), anti-CHOP (Sigma-Aldrich,), anti-BiP (Sigma-Aldrich,), anti-pro/active-CASP3/caspase 3 (Novus Biologicals) and anti-NFκB (Abcam). A secondary biotinylated anti-immunoglobulin followed by horseradish peroxidase-conjugated streptavidin (BioGenex, HK330–5K) was used according to the manufacturer's instructions. 3-amino-9-ethyl-carbazole (BioGenex, HK092–5K) in acetate buffer containing 0.05% H2O2 was used as the substrate. The sections were counterstained with hematoxylin. The primary antibody was replaced by nonimmune serum for negative control slides. Image analysis and cell counting was performed using the Nikon software, Nis Elements 3.0.
Positive signals were distinguished from cytoplasmic staining and were quantified using automated image analysis from different fields at 40X magnification. All photomicrographs were taken with the same settings for exposure time and light. The results are represented in bar graphs as the percentage of positive cells per field.
Positive signals were distinguished from cytoplasmic staining and were quantified using automated image analysis from different fields at 40X magnification. All photomicrographs were taken with the same settings for exposure time and light. The results are represented in bar graphs as the percentage of positive cells per field.