Polylysine coated slides

The paraffin sections were made and stained with hematoxylin and eosin (H&E) using a routine method [22 (link)] and SABC-immunohistochemistry (IHC) using the method described as follows: the samples were sectioned (4 μm) and placed on the polylysine-coated slides (molecular weight: 150,000–300,000; concentration: 0.10% (w/v) in water, Sigma, St. Louis, MO, USA). After deparaffination, enzyme-induced epitope retrieval was performed using 1.0 mg/mL trypsin 1:250 (250.N.F.U/mg, Sigma, St. Louis, MO, USA) treatment, which was followed by endogenous peroxidase blocking (3% H₂O₂). Then, blocking was performed using 5% bovine serum albumin (BSA, Boster, Wuhan, China) treatment. All samples with the primary antibody were incubated at 4 °C overnight. After being rinsed with PBS 5 min × 3 times, HRP-conjugated secondary antibody was applied for 1 h in humidified box at 37 °C. After being rinsed with PBS 5 min × 4 times, the SABC was applied for 30 min in humidified box at 37 °C. After being rinsed with PBS 5 min × 4 times, DAB Kit (ZSGB-BIO, Beijing, China) was used at room temperature for detection. Slides were counterstained with hematoxylin (Solarbio, Beijing, China) and mounted with neutral balsam (Solarbio, Beijing, China). Sections were examined using an Olympus microscope (Olympus, Hamburg, Germany).

Cells attached to polylysine-coated slides (Sigma, St. Louis, MO) were fixed (4% paraformaldehyde) and permeabilized (0.1% Triton X-100) prior to incubation with primary and fluorescent (FITC)-labeled secondary antibodies, respectively. Fluorescence was visualized in an IX70 fluorescent microscope (Olympus, Japan), equipped with a Coolsnap HQ digital camera (Photometrics, Tucson, AZ)