Horseradish peroxidase hrp conjugated secondary antibody

Paraffin-embedded tissue sections were deparaffinized at 60 °C for 20 min, cleared in xylene and subjected to a series of graded alcohols. For hematoxylin and eosin (H&E) staining, slides were stained with Mayer’s hematoxylin (Sigma-Aldrich) and 0.1% sodium bicarbonate and were counterstained with Eosin Y solution (Sigma-Aldrich). For IHC, slides were heated with saline-sodium citrate (SSC) buffer at 95–100 °C. After cooling, the slides were blocked with blocking solution (2% goat serum, 2% BSA, and 0.05% Tween in PBS) at room temperature and were incubated with a primary antibody diluted in blocking solution at 4 °C. Endogenous peroxidase activity was quenched with 0.3% H₂O₂. Slides were incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody (GeneTech) at room temperature, developed with a 3,3′-diaminobenzidine (DAB) substrate (GeneTech), counterstained with hematoxylin, and dehydrated with a series of graded alcohols. The positive-staining density was measured by a computerized imaging system composed of a Leica charge-coupled device (CCD) DFC420 camera connected to a Leica DM IRE2 microscope (Leica Microsystems Imaging Solutions Ltd). The H-score system was utilized, which evaluated staining intensity (0 to 3) and the percentage of positively stained cells (0 to 100), with a final score ranging from 0 to 300.

IHC was performed as previously described [13 (link)]. Immunostaining was conducted using the primary antibodies against IL-18 (1 : 200, Cell Signaling Technology (CST)), Ki-67 (1 : 400, CST), and cleaved caspase-3 (1 : 400, CST). Then, the tissue sections were incubated with horseradish peroxidase- (HRP-) conjugated secondary antibody (GeneTech, Shanghai, China). For IL-18 and cleaved caspase-3, the staining intensity (SI) was scored as zero (negative), one (weak), two (moderate), or three (strong) and the staining extent (SE) was scored as zero (0%), one (1%–25%), two (26%–50%), three (51%–75%), or four (76%–100%). The overall staining score (OSC) was calculated by the product of the SI score and SE score. According to the OSC, specimens were divided into different groups as follows: the negative expression (0–4), weak expression (5–8), and strong expression (9–12) groups. For Ki-67, patients were also grouped by different expression: the low-expression group (nuclear staining tumor cells < 46%) and high-expression group (nuclear staining tumor cells ≥ 46%).