3 3 diaminobenzidine dab stain

Liver tissues from human liver cancer patients, Tg^TM4SF5 FVB/N mice, or C57BL/6 (WT or Tm4sf5^−/− KO) mice treated with or without DEN and in the absence or presence of TSAHC were processed for immunohistochemistry. Liver sections were fixed with 3.7% formaldehyde and embedded in paraffin. The fixed liver sections were deparaffinized and rehydrated. Antigen retrieval was performed with heat-induced epitope retrieval (HIER) using sodium citrate buffer (pH 6.0). Quenching and blocking were performed using 3% H₂O₂ in distilled water and 1% normal goat serum in phosphate-buffered saline (PBS). Antigens were stained using the avidin–biotin complex (ABC) method (VECTASTAIN Elite ABC HRP Kit, Vector) and were detected using 3,3′-diaminobenzidine (DAB) stain (Vector). Antibodies against TM4SF5 [17 (link)], collagen I (Acris Antibodies), pY⁷⁰⁵STAT3 (Cell Signaling Technology), normal rabbit or mouse IgG, α-SMA (Sigma-Aldrich), α-fetoprotein (AFP), CD34, Ki67, laminins (Abcam), α-l-fucosidase [FUCA (AFU)], and laminin γ2 (Santa Cruz Biotechnology) were used for immunostaining. Ten random images per slide were saved using a digital slide scanner (MoticEasyScan, Motic, British Columbia, Canada). The tissues were also processed with Masson’s trichrome as well as hematoxylin and eosin stains, as previously described [27 (link)].

Mice were sacrificed by cervical dislocation and lungs were harvested, fixed overnight in 4% paraformaldehyde, and paraffin-embedded. Lung tissue sections were dewaxed and rehydrated, followed by antigen retrieval in Tris-EDTA buffer (pH 9.0). Sections were then blocked using 5% goat serum and incubated with 1:100 dilution of the following antibodies overnight at 4 °C: Glut1 (ProteinTech, Rosemont, IL, USA, Cat. No. 21829-1-AP) Hk2 (Cell Signaling, Danvers, MA, USA, Cat. No. 2867S), and Pkm2 (Cell Signaling, Cat. No. 4053S). Sections were subsequently incubated with 1:500 dilution of an horseradish peroxidase (HRP)-conjugated anti-rabbit antibody (Invitrogen, Cat. No. 32260) for one hour at room temperature. Expression was detected using the 3,3′-diaminobenzidine (DAB) stain (Vector Laboratories, Berlingame, CA, USA) and counterstained with hematoxylin. Hematoxylin and eosin staining was performed by the University of Louisville’s Special Procedures Laboratory within the Department of Pathology. Imaging was performed using an Aperioscope digital slide scanner (Leica Biosystems, Buffalo Grove, IL, USA).