Immunohistochemical Staining of Gal1 in Melanoma

Formalin-fixed, paraffin-embedded sections were deparaffinized during 18 h in xylene followed by rehydration essentially as described (34 (link)). Heat-mediated antigen retrieval was performed using citrate-based retrieval buffer (10 mM). Samples were blocked in peroxide blocking buffer (3% H₂O₂), followed by blocking buffer (PBS; 1% BSA and 5% normal serum) during 1 h at room temperature. Samples were incubated with HMB45 + M2-7C10 + M2-9E3 anti-melanoma cells Ab (ab732; Abcam) or with anti-Gal1 rabbit polyclonal Ab at 4 °C overnight in a humidified chamber. Then, samples were washed and incubated with a biotinylated secondary Ab (ABC Elite Kit; Vector Laboratories) followed by streptavidin–alkaline phosphatase incubation (ABC-AP Kit; Vector Laboratories). Samples were then washed in PBS, incubated in red chromogen (Vector Red), and counterstained with Mayer’s hematoxylin. The Gal1 score was calculated by a semiquantitative assessment of both the intensity of staining (graded as 0 to 3 using adjacent normal parenchyma as reference) and the percentage of positive cells (graded as 0 to 5). Expression levels were categorized as low/medium or high according to the median value of the score.