Mach 2 double stain 1 kit

Paraffin‐embedded sections containing formalin‐fixed LSCC specimens were deparaffinized and rehydrated. Target Retrieval Solution (Dako) was used to retrieve antigens. GSTO2 immunoactivity was examined using anti–GSTO2 antibody (HPA048141, Sigma‐Aldrich), an ImmPACT DAB Peroxidase Substrate Kit (Vector Laboratories), and hematoxylin for counterstaining. Mouse anti–cytokeratin 5/6 (CK5/6) antibody (D5/16B4, Cell Marque) was used for double staining with GSTO2. Bound antibodies were detected using Alexa Fluor 488‐conjugated goat anti–mouse IgG (for CK5/6) or TRITC‐conjugated goat anti–rabbit IgG (for GSTO2). For the double staining with GSTO2, a MACH 2 Double Stain 1 Kit (Biocare Medical) and a Warp Red Chromogen Kit (Biocare Medical) were used to detect anti–CC16 (AY1E6, Hycult Biotech) and anti–surfactant protein A (SP‐A; PE10, Gene Tex) signals.

Formalin-fixed, paraffin-embedded sections of surgical specimens from patients with ESCC were deparaffinized and rehydrated. The antigen was retrieved using 10 mM sodium citrate buffer (pH 6.0) in an autoclave for 30 minutes at 95°C. Sections were stained using an anti-CSTA antibody (HPA001031; Sigma-Aldrich, Inc.). Diaminobenzidine staining was performed using the ImmPACT DAB Peroxidase Substrate Kit (Vector Laboratories, Burlingame, CA), and counterstaining was performed using hematoxylin. For double staining of CSTA and Ki67, the MACH 2 Double Stain 1 Kit (Biocare Medical, Concord, CA) and Warp Red Chromogen Kit (Biocare Medical) were used to detect anti-Ki67 antibody (MIB-1, Dako, Glostrup, Denmark) signals. All slides were reviewed by 2 observers who were blind to clinical and pathological data. On the basis of the proportion of CSTA-positive areas in the malignant tissue, we classified ESCC cases into CSTA-negative (the percentage of CSTA-positive tumor cells was ≤20% on immunostaining) and CSTA-positive groups (the percentage of CSTA-positive tumor cells was >20%). Because there was a clear difference between CSTA-positive/negative, there appeared to be no difficulty judging CSTA-positive/negative. Intraobserver reliability of the CSTA staining results showed no statistically significant differences. The 2 observers exhibited high interobserver reliability.

Formalin-fixed paraffin-embedded sections of surgically resected LSCC specimens were deparaffinized and rehydrated. Target Retrieval Solution (Dako, Glostrup, Denmark) was used to retrieve the antigens. Sections were stained with anti-KLK13 antibody (Sigma-Aldrich, Inc., St. Louis, MO, USA) and an ImmPACTTM DAB peroxidase Substrate Kit (Vector Laboratories, Burlingame, CA, USA), and counterstaining was performed using hematoxylin. Anti-human papillomavirus (HPV) type 16 L1 antibody (GeneTex Inc. CA, USA) was used to examine the HPV status of LSCC patients. For double staining with KLK13, the MACH 2 Double Stain 1 Kit (Biocare Medical, Concord, CA) and Warp Red Chromogen Kit (Biocare Medical) were used to detect the anti-p40 (Biocare Medical) or anti-E-cadherin antibody (Santa Cruz Biotechnology, Inc., Dallas, TX) signals. Regarding the expression of E-cadherin, we classified the LSCC cases into E-cadherin-positive and -negative groups based on the localization of E-cadherin at the membranes of tumor cells in dominant area. Two observers reviewed all slides, without any access to clinical or pathological data, exhibiting high inter-observer reliability with a clear difference between KLK13-positive/negative samples.