Chemmate hematoxylin

Following deparaffinization and quenching of endogenous peroxidase, sections were incubated with 1% bovine serum albumin in PBS. Subsequently, the slides were treated with rabbit primary antibodies against OBR, p-STAT3, CEBPD, and MCL1 followed by incubation with goat anti-rabbit IgG antibodies. The slides were counterstained with ChemMate Hematoxylin (Dako Cytomation) and mounted and observed under a BX51 microscope (Olympus). Sections were semiquantitatively scored twice for the extent of immunoreaction as follows. For the first score, positive cells in less than 5% of the total indicated a score of 0; positive cells in 5%–25% indicated a score of 1; positive cells in 25%–50% indicated a score of 2; and positive cells in greater than 50% of the total indicated a score of 3. For the second score, no coloration was scored as 0; pale yellow was scored as 1; yellow was scored as 2; and brown was scored as 3. The 2 scores were then multiplied; the resulting combined scores were categorized as follows: a score of 0 was negative (–); scores of 1–3 indicated weak immunoreactivity (+); scores of 4–6 indicated moderate immunoreactivity (++); and scores of 7–9 indicated strong immunoreactivity (+++).

After the tissues were sections, deparaffinized and quenched to block endogenous peroxidase activity, they were incubated in 1% bovine serum albumin (BSA) in PBS. The slides were subsequently incubated first with rabbit anti-human-TCTP antibodies (1:500, CST) and then with goat anti-rabbit IgG antibodies. The slides were counterstained with ChemMate Hematoxylin (DakoCytomation) and mounted and observed under a microscope (Olympus). The TCTP expression via IHC was blinded read by two pathologists independently. The sections were semi-quantitatively scored according to the amount and level of immunoreactivity, as follows: 0 (less than 5% positive cells), 1 (5–25% positive cells), 2 (25–50% positive cells), and 3 (greater than 50% positive cells); and 0 (no coloration); 1 (pale yellow); 2 (yellow); and 3 (reddish-brown). The two scores were then multiplied to achieve the following composite scores: 0, negative (−); 1–3, weak (+); 4–6, moderate (++); and 7–9, strong (+++).