Meyer s hematoxylin

Deparaffined sections were stained for PAS using PAS-Staining Kit (Sigma-Aldrich). In brief, the slides were treated for 5 min in periodic acid solution, rinsed twice with distilled water, 15 min in Schiff’s reagent, 2 rinses, 15 min in Meyer’s hematoxylin (Vector laboratories) solution, 2 rinses, and serial dehydration in 70%, 80%, 90%, 100% ethanol before putting on the coverslip.

4-μm thick sections were cut from the paraffin blocks and immunostained as described [10 (link)]. Sections were deparaffinized in xylene, and hydrated in descending grades of ethanol. Sections were incubated with 3% hydrogen peroxide to inactivate endogenous peroxidase activities. After blocking in 1% normal goat serum (#S-1000, Vector Laboratories, Inc.) for 1 h, the sections were incubated with primary antibodies against phospho-EGFR (1:100) overnight at 4 °C, followed by incubation with biotinylated anti-rabbit IgG (#BA-1000, Vector Laboratories, Inc.) for 30 min. The sections were then incubated with horseradish peroxidase streptavidin (#SA-5004, Vector Laboratories, Inc.) for 30 min. The signal was developed using the 3,3′ diaminobenzidine tetrahydrochloride (DAB) kit (#SK-4100, Vector Laboratories, Inc.) and counterstained with Meyer’s hematoxylin (#H-3404, Vector Laboratories, Inc.). The staining intensity of the phosphorylated EGFR on the cell membrane was manually scored. An intensity scale ranging from 0 for no staining to 3+ for the most intense staining is used. H-Score is calculated using the following formula: H − Score = (%at 0) × 0 + (%at 1+) × 1 + (%at 2+) × 2 + (%at 3+) × 3.

Immunohistochemistry was performed as described previously with minor modifications [23 (link)]. Briefly, tissue slides printed with normal lung or lung cancer tissues were purchased from SuperBioChips Laboratories (Seoul, Korea). The slides were incubated first with mouse anti-VCAM-1 monoclonal antibody (1:200; Abcam, Cambridge, MA, USA) and then with biotinylated goat anti-mouse IgG (1:200; Vector Laboratories, Burlingame, CA, USA). Immunoreactive proteins were visualized using VECTASTAIN ABC Reagent (Vector Laboratories). For chromogenic reactions, the slides were incubated with fresh 3,3′-diaminobenzidine tetrahydrochloride solution (Vector Laboratories). All samples were counterstained with Meyer’s hematoxylin (Vector Laboratories). VCAM-1 expression was observed by light microscopy using an Olympus BX51 microscope (Olympus, Tokyo, Japan), and RGB images were acquired using Paint Shop Pro X software (Corel, Ottawa, ON, Canada). After performing background subtraction, VCAM-1 expression was quantified by measuring the signal density with Image J software version 1.48v (National Institutes of Health, Bethesda, MA, USA).