Immpact vip

The primary eye and liver FFPE tissues were deparaffinized and hydrated followed by blocking endogenous peroxidase activity in 3% H₂O₂ for 10 min. Antigen retrieval was completed with 10 mM citrate buffer in a microwave for 3 min. Samples were cooled down and washed with water followed by blocking with Biocare blocking reagent for 10 min (#BS966M, Biocare, Pacheco, CA, USA). Tissues were incubated with primary antibody at 1:100 dilution for 1 h at room temperature, followed by 5 min buffer wash. The slides were incubated with Envision plus labeled polymer, anti-rabbit-HRP (#K4011, Dako, Santa Clara, CA, USA) for 30 min at room temperature, followed by 5 min buffer wash. Next, the slides were incubated with ImmPACT VIP (#SK-4605, Vector Labs, Newark, CA, USA) (this is a purple stain) for about 3–10 min. Finally, the slides were washed, counterstained, dehydrated, and prepared for viewing.

IHC was performed on 4-μm-thick sections using ABC Vectastain kit (Vector Labs) with antibodies to PKM1 (1:500, CST 7067), PKM2 (1:500, CST 4053), and phospho-Histone H3 (1:200, CST 9701). The staining was visualized with DAB (Vector Labs, SK-4100), and the slides were counterstained with hematoxylin. For dual staining, DAB and ImmPACT VIP (Vector Labs, SK-4605) were used. Hematoxylin and eosin (H&E) staining was performed on a Varistain Gemini automated slide stainer (Thermo Shandon). Soft tissue sarcoma human tissue array, SO2081, was purchased from US Biomax Inc. (Rockville, MD USA). The TMA was scored for PKM1 and PKM2 intensity. Tumors that showed no positive staining were given a score of 0, those with weak staining were given a score of 1, those with strong positive staining were given a score of 2, and those with very strong staining were given a score of 3.
ImageJ software was used for quantification of pHH3 staining, and four to five 10× fields were counted per tumor.