Dp71 optical microscope

Mounted sections were submerged in sodium citrate buffer (0.1 M, pH 6) and left in a water bath for 15 min to expose antigen epitopes. This was followed by the incubation of the sections with 0.3% H₂O₂ in methanol to prevent any nonspecific binding to endogenous peroxidase. Rabbit anti-rat primary antibodies were added to the sections and incubated overnight at 4 °C. Rabbit monoclonal antibodies against Bax [E63] (ab32503), β-catenin [E247] (ab32572,) COX2 [EPR12012] (ab179800), Ki67 antibody (ab15580), and NF-κB-p65 (ab16502) were purchased from Abcam (Cambridge, CB2 0AX, UK), while anti-PCNA and anti-caspase-3 were purchased from Cell Signaling Technology Inc. (Danvers, MA, USA). Next, the slides were rinsed with PBS and subjected to incubation with the secondary antibody polyvalent biotinylated goat-anti-rabbit for 10 min at room temperature (1:200 dilution). The standard staining protocol was conducted using the Universal LSAB kit and DAB plus substrate kit. Additional counter-staining was carried out using hematoxylin. The slides were examined and photographed under an Olympus DP71 optical microscope. Ten fields were then chosen at random for the quantification of positive cells in individual samples (×400).

Mounted sections were immersed in sodium citrate buffer (0.1 M, pH 6) and placed in a water bath for 15 min to unmask antigen epitopes. Afterwards, sections were incubated with 0.3% H₂O₂ in methanol to block nonspecific binding to endogenous peroxidase. Sections were incubated overnight at 4°C with rabbit anti-rat primary antibodies, anti-COX-2, anti-iNOS, anti-NF-_kB-P65, and anti-Ki-67; in addition to M30 CytoDeath, monoclonal ED-2 anti-rat antibody, and polyclonal anti-rabbit antibodies, anti- GST-p and anti-p-TNFR. After incubation, slides were washed with PBS and incubated with polyvalent biotinylated goat-anti-rabbit, a secondary antibody, for 10 min at room temperature (1:200 dilution). Universal LSAB kit and DAB plus substrate kit were both used to perform a standard staining protocol. Hematoxylin was used in additional counter-staining. Slides were observed under an Olympus DP71 optical microscope, and tissue images were obtained.