Ab50692

Immunohistochemistry for AUF1 was performed on formalin-fixed paraffin-embedded tissues using anti-AUF1 antibody from Abcam (ab50692) overnight at a dilution of 1:500, and slides were stained using automated staining platform (Ventana). Envision + polymer (ready to use; Dako) was used as a secondary antibody. Color was developed with 3,3′-diaminobenzidine (DAB), and instant hematoxylin (Shandon) was used for counterstaining. The AUF1 level was evaluated and verified by two qualified pathologists, who scored both the proportion of positive cells and the intensity of AUF1 expression in both cancer cells and their stromal fibroblasts, and an immunoreactivity score was determined and used for statistical analysis.

For CLSM, 5 × 10⁴ OSCC cells (SCC4/MDA1986) were plated on cover slips and grown for 24 h fixed in acetone: methanol mixture (1:1) at −20 °C for 20 min. [28 (link)]. Cells were permeabilized with PBS-0.1 % Tween 20, non-specific binding blocked with 5 % BSA for 1 h; cells were incubated with rabbit polyclonal hnRNPD (ab50692)/mouse monoclonal hnRNPK (ab23644, Abcam, CA) antibody at 4 °C overnight. Expression of proteins was determined using fluorescein isothiocyanate (FITC)/TRITC-labeled secondary antibodies (DAKO Cytomation, Denmark) [27 (link)].

Paraffin-embedded tissue sections were deparaffinized, antigen was retrieved, endogenous peroxidase activity was quenched with hydrogen peroxide (0.3 % v/v) and non-specific binding blocked with 1 % bovine serum albumin (BSA). The slides were incubated with either rabbit polyclonal anti-hnRNPD antibody (1 μg/ml, ab50692, Abcam, CA) or mouse monoclonal anti-hnRNPK antibody (ab23644) or rabbit polyclonal 14-3-3ζ antibody (sc-1019) for 16 h at 4 °C. The primary antibody was detected using the Dako Envision kit (Dako CYTOMATION, Glostrup, Denmark) with diaminobenzidine as the chromogen and counterstained with hematoxylin [26 (link), 27 (link)]. The sections were evaluated by light microscopy and scored using a semi-quantitative scoring system for both staining intensity (nuclear/cytoplasmic) and percentage positivity as described earlier [26 (link), 27 (link)]. The tissue sections were scored based on the % of immunostained cells as: 0–10 % = 0; >10–30 % = 1; >30–50 % = 2; >50–70 % = 3 and >70–100 % = 4. Sections were also scored semi-quantitatively on the basis of staining intensity as negative = 0; mild = 1; moderate = 2; intense = 3. Finally, a total score was obtained by adding the score of percentage positivity and intensity giving a score range from 0 to 7. IHC score thus obtained for different proteins were subjected to statistical analysis.