Ab210959

In brief, paraffin sections of HPSCC tumors were deparaffinized, hydrated, and antigen retrieval was performed at 100 °C in citrate buffer (pH 6.0) for 20 min. After cooling to 25 ℃, the sections were incubated with SPRR3 (Proteintech, 11742-1-AP), Ki67 (Origene, TA802544), LAMC2 (Abcam, ab210959), and fibroblast activation protein (FAP, Abcam, ab207178) antibodies overnight at 4 °C, followed by incubation with the goat anti-rabbit/mouse IgG-HRP polymer (Proteintech, PK10006) for 1 h at 25 ℃. Sections were then developed using a diaminobenzidine chromogenic solution, counterstained using hematoxylin, dehydrated using ethanol, cleaned with xylene, and mounted.
To quantify the target protein expression, the percentage of positive cells and the staining intensity were determined in five randomly selected fields using a microscope at 400 × magnification. For this, the cells were counted by two independent pathologists, neither of whom had knowledge or information regarding the clinical status of the patients. Scoring was conducted as previously described [27 (link)]; scores ≥ 2 points were considered positive expression.
To explore the relationship between the target protein and patient prognosis, survival analysis was performed in the R package “survival”, and the Survfit function was used to model the Kaplan–Meier survival curve.

HNSCC samples and normal samples were collected from humans for IHC; the methods for IHC are described in our previous study 19 (link). IHC was performed with anti-PLAU (17968-1-AP, Proteintech, 1:500) and anti-LAMC2 (ab210959, Abcam, 1:500) antibodies. The methods of immunofluorescence staining of PLAU and LAMC2 were similar to those of IHC. Sections were subjected to rehydration and antigen retrieval, followed by elimination of autofluorescence. Primary antibodies were then applied and incubated at 4°C overnight, followed by fluorescence-conjugated secondary antibody incubation and DAPI staining of nuclei.