Envision target retrieval solution

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)].

Formalin-fixed, paraffin-embedded tissue sections (5 μm) were deparaffinized using routine techniques, and placed in 200 ml of EnVision™ target retrieval solution (pH 6.0; Dako, Hamburg, Germany) for 20 minutes at 100°C. After cooling for 20 minutes, slides were quenched with 3% H₂O₂ for 5 minutes before incubating with primary antibodies against cleaved caspase 3 (#cat 9661; 1:200; Cell Signaling, Danvers, MA, USA) to detect apoptotic cells or marker of proliferation Ki-67 (#cat M724029-2, 1:25, Dako) using a Dako Autostainer (Dakocytomation). Immunostaining was visualized using the EnVision™+ kit (Dako).

Serial sections were cut from the selected FFPE tumor blocks (N = 129) and mounted on FLEX IHC Microscope Slides (K8020, DAKO, Glostrup, Denmark). The pretreatment processes were performed using PT Link (DAKO). Heat-induced epitope retrieval was achieved with Envision Target Retrieval Solution (DAKO) at pH 9 and 97 °C for 20 min.
Staining was performed using a DAKO Autostainer Link 48 (DAKO).
Endogenous peroxidase activity was blocked by Envision FLEX Peroxidase-Blocking Reagent (DAKO). The primary antibodies were mouse monoclonal anti-CD3 (code M7254, DAKO) diluted 1:600, and anti-CD8 (code M7103, DAKO) diluted 1:300. The primary antibodies were diluted with Envision Flex antibody diluent (code S2022 DAKO).
Primary antibodies were incubated for 30 min at room temperature, and for amplification Envision Flex + Mouse(Linker) (DAKO) was used for 20 min. Bound antibodies were detected using Envision FLEX/HRP (DAKO) and visualized by Envision FLEX DAB (DAKO) and chromogene diluted in Envision Flex Substrate Buffer (DAKO). To enhance the immunohistochemical stains, the sections were incubated in 0.5% CuSO₄ in TBS buffer pH 7.6 for 10 min. Meyer’s hematoxylin (Merck, Damstadt, Germany) was used as counterstain, and finally, the histological slides were coverslipped with Tissue-Tek PERTEX (Histolab Products AB, Göteborg, Sweden).

Serial sections were cut from the selected tumour blocks (N = 129) and mounted on FLEX IHC Microscope Slides (K8020, Agilent DAKO products, Glostrup, Denmark). The pretreatment processes were performed using PT Link (DAKO). Heat-induced epitope retrieval was achieved with Envision Target Retrieval Solution (DAKO) at pH 9 and 97 °C for 20 min. Staining was performed using a DAKO Autostainer Link 48 (DAKO). Endogenous peroxidase activity was blocked by Envision FLEX Peroxidase-Blocking Reagent (DAKO). The primary antibody was mouse monoclonal cytokeratin AE1/AE3 (code M3515, DAKO) diluted 1:250 with Envision Flex antibody diluent (code S2022 DAKO). Primary antibody was incubated for 30 min. at room temperature, and for amplification Envision Flex+ Mouse (Linker) (DAKO) was used for 20 min. Bound antibodies were detected by Envision FLEX/HRP (DAKO) and visualised by Envision FLEX DAB (DAKO) with chromogen diluted in Envision Flex Substrate Buffer (DAKO). Meyer’s hematoxylin (Merck, Darmstadt, Germany) was used as counterstain and finally, the histological slides were cover slipped with Tissue-Tek PERTEX (Histolab Products AB, Göteborg, Sweden).

Formalin-fixed, paraffin-embedded xenograft tumor sections (5 µm) were deparaffinized using routine techniques, and placed in 200 mL of EnVisionTM target retrieval solution (pH 6.0; Dako, Hamburg Germany) for 20 min at 100 °C. After cooling for 20 min, slides were quenched with 3% H₂O₂ for 5 min before incubating with a primary antibody against cleaved caspase 3 (Abcam, ab2302, 1:500, Cambridge, Storbritannien) to detect apoptotic cells using a Dako Autostainer. Immunostaining was visualized using the EnVisonTM+ kit (Agilent Dako, Santa Clara, CA, USA). In addition, slides were also stained with hematoxylin and eosin.

Formalin-fixed, paraffin-embedded lung sections (5 µm) were deparaffinized using routine techniques and placed in 200 mL of EnVisionTM target retrieval solution (pH 6.0; Dako, Hamburg Germany) for 20 min at 100 °C. After cooling for 20 min, slides were quenched with 3% H₂O₂ for 5 min. Immunostaining was visualized using the EnVisonTM + kit (Dako). In addition, slides were also stained with hematoxylin and eosin.