Journal: Histochemistry and Cell Biology
Article Title: Immunohistochemical analysis to detect a molecular signature in intervertebral disc degeneration
doi: 10.1007/s00418-025-02434-w
Figure Lengend Snippet: Analysis of FOXO3a, SOD2, HIF1α, GLUT1, and Bry expression in IVD tissues with Pfirrmann grade III. Protein levels were assessed by immunohistochemistry and quantified by densitometric analysis of immunostaining using ImageJ software. Results were expressed as the percentage of positive cells per area, based on five representative sections per sample ( n = 40, Pfirrmann grade III). Data are presented as box-and-whisker plots, displaying min to max (the line indicates the median). Comparisons were made on the basis of the following parameters: a sex (female, male), age (18–40, 41–60, > 60 years), smoking status (non-smoker [NS], former smoker [FS], current smoker [CS]), and body mass index (BMI) (normal weight [NW], overweight [OW], obese [OB]); b anatomical site of surgery (L2–L3, L3–L4, L4–L5, L5–S1), duration of symptoms before surgery (< 6 months, ≥ 6 months), and area of infiltration (moderate, abundant)
Article Snippet: For immunohistochemical evaluation, sections were incubated overnight (4 °C) with a primary antibody against FOXO3a (#ab70315, rabbit anti-human, 1:100 dilution; Abcam, Cambridge, UK), SOD2 (#sc-133134, mouse anti-human, 1:100 dilution; Santa Cruz biotech., Dallas, USA), HIF1α (clone H1alpha67, mouse anti-human, 1:200 dilution; Novusbio, Centennial, USA), GLUT1 (#TA301678, mouse anti-human, 1:200 dilution; Origene, Rockville, USA), and BRY (#ab209665, rabbit anti-human, 1:500 dilution; Abcam), followed by treatment with Vectastain ABC solution (#MP-7500; Vectorlabs, Burlingame, USA) for 30 min. To achieve specificity, the working concentrations of primary antibodies were selected by following the dilution guidelines and technical specifications provided by the antibody’s manufacturer.
Techniques: Expressing, Immunohistochemistry, Immunostaining, Software, Whisker Assay