Micropolymer hrp conjugated secondary antibody

Tumors were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned at 3 μM thickness. Paraffin-embedded tumor sections were deparaffinized, rehydrated, and incubated with citrate buffer (ImmunoBioScience, Mukilteo, WA, USA) 95 °C for antigen retrieval. To quench endogenous peroxidase activity, the slides were incubated in 1.4% hydrogen peroxide/methanol. After blocking with 2.5% normal horse serum (Vector Laboratories) for 1 h, the slides were incubated with anti-cleaved Caspase3 antibody (Cell signaling) at 4 °C overnight. After PBS washing three times, the slides were incubated with a micropolymer HRP-conjugated secondary antibody (Vector Laboratories) for 1 h at room temperature. Antigens were demonstrated by DAB (Vector Laboratories). The slides were counterstained with EASYSTAIN Harris Hematocylin (YD diagnostics, Yongin, Korea). Finally, the slides were dehydrated in graded ethanol and mounted with Permount mounting medium (Fisher Scientific, Waltham, MA, USA). The images were acquired with Axio Scan.Z1 (ZEISS, Oberkochen, Germany).

Tumors were fixed in 4% paraformaldehyde and embedded in paraffin. Paraffin‐embedded tumors were sectioned at 3 µm thickness and section slides were deparaffinized, rehydrated, and incubated with citrate buffer (ImmunoBioScience, AR‐6544‐05) at 95 °C for antigen retrieval. The slides were incubated in 1.4% hydrogen peroxide/methanol for quenching endogenous peroxidase activity and blocked with 2.5% normal horse serum (Vector Laboratories, MP‐7401) for 1 h. The slides were incubated with anti‐cleaved Caspase 3 (Cell signaling, 9664S) or anti‐HIF1α antibodies (Cayman, 10006421) at 4 °C overnight. The following day, the slides were incubated with a micropolymer HRP‐conjugated secondary antibody (Vector Laboratories, MP‐7401) for 1 h at room temperature. Antigens were revealed by DAB (Vector Laboratories, SK‐4100) and then the slides were counterstained with EASYSTAIN Harris Hematocylin (YD diagnostics, S2‐5). Finally, the slides were dehydrated in increasing concentrations of ethanol, mounted with Permount mounting medium (Fisher Scientific, SP 15–100), and imaged using Axio Scan.Z1 (ZEISS).