Cy5 conjugated goat anti rabbit igg

Human NP cells were seeded on coverslips, exposed to 50 ng/ml TNF-ɑ, and treated with or without 50 nM THZ1, 4% paraformaldehyde was used to fix cells for 10 min, 0.1% Triton X-100 permeabilization for 10 min, 10% goat serum block for 1 h, and PBS was used to wash cells three times at intervals before incubation with MMP13 (1:3,000 dilution; ab39012; Abcam), Pol II Ser5P (1:1,000 dilution; ab5408; Abcam) at 4°C overnight. After washing three times with PBS, samples were incubated with Cy5-conjugated goat anti-rabbit IgG or goat anti-mouse IgG (1:200; Abcam) in PBS for 1 h, and then stained with DAPI for 10 min at room temperature (Life Technologies). Fluorescence signals were imaged using a fluorescence microscope (Olympus IX71; Olympus).

TMA construction were performed as described previously with refs. [21 (link), 31 (link)]. The TMA glass slides were cleaned with PBS and fixed in 4% paraformaldehyde for 10 min. After 1 h of blocking using 5% BSA (Amresco, OH, USA), the slides were incubated with anti-OTUB2, anti-KRT80, and anti-Ki67 (1:100), accompanied by goat anti-rabbit-Cy5-conjugated IgG (1:100, ZSGB-BIO) (1:100, Abcam) or goat anti-mouse-TRITC-conjugated IgG (1:100, ZSGB-BIO). Following washing, the slides were examined using an automated quantitative pathology imaging system (PerkinElmer, USA). The IF score was determined using the extent and intensity of staining (extent: 0 = no staining, 1 = 0–10%, 2 = 10–50%, and 3 = 50–100%; and intensity: 0 = negative, 1 = weak, 2 = moderate, 3 = solid). The total score was determined as the multiplication of the intensity and extent scores. 0–3 were deemed to be negative expressions. (low); 4–5 were deemed to be a weak expression (low); and 6–12 were considered to be a strong expression (high). The IF score was determined separately by two pathologists who were blinded to the patient characteristics.

The cells were grown on glass slides, washed with PBS, and fixed in 4% paraformaldehyde for 10 min. After 1 h blocking with 5% BSA (Amresco, Solon, OH, USA), the slides were incubated with anti-S100A14 (1:100, Proteintech) and anti-Vinculin (1:100, Proteintech) followed by goat anti-mouse-TRITC-conjugated IgG (1:100, ZSGB-BIO) or goat anti-rabbit-Cy5-conjugated IgG (1:100, Abcam). After the slides were washed, they were studied using a confocal fluorescence imaging microscope (LSM780, ZEISS, Göttingen, Germany).