Signal stain boost immunohistochemical detection reagent

Endometrial tumor slides (4 µm) from LKB1^fl/fl p53^fl/fl mice were first incubated with protein block solution (Dako) for one hour and then added with the primary antibodies overnight in a cold room. The slides were then washed with TBS-T and incubated with appropriate secondary antibodies at room temperature for one hour. After removing the secondary antibody, the specific staining was visualized using the Signal Stain Boost Immunohistochemical Detection Reagent (Cell Signaling Technology, Danvers, MA), according to the manufacturer’s instructions. Individual slides were scanned using the Motic scanner, and digital images were analyzed for target protein expression using ImagePro software (Vista, CA).

The mouse tumor tissue was formalin-fixed and paraffin-embedded. Slides (5 μm) were first incubated with protein block solution (Dako) for 1 hour and then with the primary antibodies for Ki-67 (1:400), phosphorylated-S6 (1:300) and phosphorylated-AMPK (1:100) for 2 hours at room temperature. The slides were then washed and incubated with appropriate secondary antibodies at room temperature for 1 hour. The slides were washed, and the specific staining was visualized using the Signal Stain Boost Immunohistochemical Detection Reagent (Cell Signaling Technology), according to the manufacturer’s instructions. Individual slides were scanned using the Aperio™ ScanScope (Aperio Technologies, Vista, CA), and digital images were analyzed for target protein expression using Aperio™.

Endometrial tumor slides (4 µm) from the Lkb1^fl/flp53^fl/fl mice were used for the IHC study. Following deparaffinization and rehydration, endogenous peroxidase was blocked in 3% H₂O₂ for 10 min. Antigen retrieval was performed in a microwave oven using a citrate buffer (sodium citrate, pH 6.0). The slides were incubated with protein block solution (Dako) for 1 h, and then the primary antibodies were added overnight in a cold room (six slides per group). The slides were then washed and incubated with appropriate secondary antibodies at room temperature for 1 h. After removing the secondary antibody, the specific staining was visualized using the Signal Stain Boost Immunohistochemical Detection Reagent (Cell Signaling Technology, Danvers, MA, USA), according to the manufacturer’s instructions. Individual slides were scanned using Motic (Feasterville, PA, USA), and digital images were analyzed for target protein expression using ImagePro software (V2, Vista, CA, USA). The IHC scoring formula was used to express the expression level of the target protein.