In Situ Hybridization of Melanoma

Antisense single-stranded DNA probe (Additional file 6: Table S5) was synthesized and end-labelled with digoxigenin (DIG) (Roche). ISH or FISH was performed in formalin-fixed paraffin-embedded melanoma sections or slides covered with cultured melanoma cells. The pre-hybridization, hybridization, anti-DIG-HRP IgG fraction monoclonal (Jackson, 200-032-156) incubation (1:200) and stained with DAB (Servicebio, G1211) was performed as described in previous studies. Stained ISH or FISH sections were imaged with a ZEISS Axio Vert.A1 microscope and at least 10 representative images were collected for statistical analysis. The ISH or FISH staining was performed “blind” with respect to the different treatments [44 (link)]. Co-localization of LINP1 with eIF2α in cSCC cells was detected using FISH for LINP1 and immunofluorescence staining for eIF2α and observed by confocal microscope.