Pt link pre treatment module for tissue specimens

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 rabbit polyclonal anti-ENO1 antibody (dilution 1:500) 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 °C in PT Link Pre-Treatment Module for Tissue Specimens (DAKO). Liquid Permanent Red (Agilent DAKO, Santa Clara, CA, USA) was utilized as a detection system. Human breast and pancreatic adenocarcinomas were stained as positive controls. 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 ENO1 immunostains was performed using the H-score [(percentage at 1+) × 1 + (percentage at 2+) × 2 + (percentage at 3+) × 3], which integrates the intensity and percentage of positive cells into a combined score. The median H-score (200) was used as a cut-off value for high (H-score > 200) and low ENO1 (H-score ≤ 200) expression [23 (link)].

Immunohistochemical studies were performed on 5-micrometer-thick tissue sections with primary antibodies against PARP1 (clone: sc-74470 (B10), dilution: 1:50, Santa Cruz Biotechnology, Santa Cruz, CA, USA), PD-L1 (E1L3N, 1:200, Cell Signaling Technology, Danvers, MA), and IDO1 (1F8.2, 1:400, Millipore, Burlington, MA) for 169, 174, and 159 primary tumors, respectively. For PARP1 and IDO1, the slides underwent heat-induced epitope retrieval (HIER) with EnVision Target Retrieval Solution (Agilent DAKO, Santa Clara, CA) in a 30-min incubation at 97 °C in PT Link Pre-Treatment Module for Tissue Specimens (DAKO). Automated immunohistochemical staining was performed in Autostainer Link 48 (DAKO) and Liquid Permanent Red (Agilent DAKO) was utilized as a detection system. For PD-L1, the slides were incubated with a ready to use EDTA based Ph 9.0 epitope retrieval solution (Leica Microsystems, Bannockburn, IL, USA) for 20 min. Immunostaining was performed in Bond 3 automated immunostainer (Leica Microsystems). Human placenta and cutaneous melanoma were used as positive controls. The percentage of tumor cells with 5% or greater PD-L1 membranous staining of any intensity was considered positive. Scoring of IDO1 and PARP1 immunostains was done using the H-score ((percentage at 1+) × 1 + (percentage at 2+) × 2 + (percentage at 3+) × 3).

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)].