Envision flex hrp magenta chromogen

Tissue microarrays (TMAs) composed of three 1.5 mm tissue cores from each tumor were automatically constructed (TMA Grand Master, Sysmex, Warsaw, Poland). Immunohistochemical analysis was performed using mouse monoclonal anti-ABCA1 antibody (clone HJ1 (ab66217), dilution 1:200, Abcam) on 4-µm-thick paraffin sections mounted on silanized slides (Agilent DAKO, Santa Clara, CA, USA). The slides underwent automated dewaxing, rehydration, and heat-induced epitope retrieval with EnVision Target Retrieval Solution (Agilent DAKO, Santa Clara, CA, USA) for 30 min at 97 ℃ in PT Link Pre-Treatment Module for Tissue Specimens (DAKO). EnVision Flex HRP Magenta Chromogen (Agilent DAKO, Santa Clara, CA, USA) was utilized as a detection system. Human normal liver was used as a positive control. Negative controls were processed using FLEX Rabbit Negative Control, Ready-to-Use (Agilent DAKO, Santa Clara, CA, USA) in place of the primary antibody. Scoring of ABCA1 immunostaining was performed using the H-score. The score is obtained by the formula: percentage of tumoral cells with weak immunoreactivity × 1 + percentage of tumoral cells with intermediate immunoreactivity × 2 + percentage of tumoral cells with high immunoreactivity × 3, giving a range of 0 to 300. The median H-score (200) was used as a cut-off value for high (H-score > 200) and low ABCA1 (H-score ≤ 200) expression [20 (link)].

The EnVision FLEX/HRP system was used for immunohistochemistry analysis (Agilent, Santa Clara, CA). For dual immunostainings, EnVision FLEX DAB + Chromogen (Agilent) was used to obtain the brown color and EnVision FLEX HRP Magenta Chromogen was used to obtain the magenta color. Antibodies, clones, dilutions, and providers are presented in Supplementary Table 2.