Tissue sections of 4 μm, serially taken after the H&E stained sections, were
stained with mIHC using the OpalTM 7 solid Tumor Immunology Kit
(PerkinElmer, Waltham MA, USA). In order to optimize the incubation time and
concentration of antibodies the staining method was modified from the
manufacturer’s instructions, as previously done for colorectal cancer.26 (link) For
optimization, PCa tissue sections from the actual cohort were used with the aim
of allowing exposure times of 30–200 ms and a signal range of 5–30 ms. The
sections were dried overnight, heated at 60°C for two hours, deparaffinized, and
rehydrated. They were then sequentially stained using specific antibodies
directed against T-box expressed T-cells (T-bet) also known as Tbx21 expressed
on Th1-cells, CD8, CD20, FOXP3, CD68, and pan-cytokeratin. The nuclear staining
was performed with DAPI and visualization of specific antibody binding together
with different Opal fluorophores (OF) from the Opal TM 7 solid Tumor
Immunology Kit. The specific antibodies directed against T-bet were used with
OF520 (green), those against CD8 with OF570 (red), those against CD20 with OF540
(yellow), those against FOXP3 with OF620 (orange), those against CD68 with OF650
(cyan), and those against cytokeratin with OF690 (magenta). The antibody working
concentration and clones were as follows: 4 μg/ml anti-T-bet (clone 4B10:
sc-21749, Santa Cruz Biotechnology, Inc, Dallas, Texas, US), 0.12 μg/ml anti-CD8
(clone C8/144B, Dako Agilent, Santa Clara, CA, US), 4 μg/ml anti-CD20 (clone L26
ab9475, Abcam, Cambridge, UK), 0.33 μg/ml anti-FOXP3 (Tregs), 0.24 μg/ml
anti-CD68 (clone KP1 M0814, Dako Agilent), and 3.6 μg/ml anti-cytokeratin
(pan-CK) for identification of tumor epithelial cells (clone AE1/AE3 M3515, Dako
Agilent). Slides were mounted using ProLong Diamond Antifade Mountant (Thermo
Fisher, Waltham, MA, USA).
stained with mIHC using the OpalTM 7 solid Tumor Immunology Kit
(PerkinElmer, Waltham MA, USA). In order to optimize the incubation time and
concentration of antibodies the staining method was modified from the
manufacturer’s instructions, as previously done for colorectal cancer.26 (link) For
optimization, PCa tissue sections from the actual cohort were used with the aim
of allowing exposure times of 30–200 ms and a signal range of 5–30 ms. The
sections were dried overnight, heated at 60°C for two hours, deparaffinized, and
rehydrated. They were then sequentially stained using specific antibodies
directed against T-box expressed T-cells (T-bet) also known as Tbx21 expressed
on Th1-cells, CD8, CD20, FOXP3, CD68, and pan-cytokeratin. The nuclear staining
was performed with DAPI and visualization of specific antibody binding together
with different Opal fluorophores (OF) from the Opal TM 7 solid Tumor
Immunology Kit. The specific antibodies directed against T-bet were used with
OF520 (green), those against CD8 with OF570 (red), those against CD20 with OF540
(yellow), those against FOXP3 with OF620 (orange), those against CD68 with OF650
(cyan), and those against cytokeratin with OF690 (magenta). The antibody working
concentration and clones were as follows: 4 μg/ml anti-T-bet (clone 4B10:
sc-21749, Santa Cruz Biotechnology, Inc, Dallas, Texas, US), 0.12 μg/ml anti-CD8
(clone C8/144B, Dako Agilent, Santa Clara, CA, US), 4 μg/ml anti-CD20 (clone L26
ab9475, Abcam, Cambridge, UK), 0.33 μg/ml anti-FOXP3 (Tregs), 0.24 μg/ml
anti-CD68 (clone KP1 M0814, Dako Agilent), and 3.6 μg/ml anti-cytokeratin
(pan-CK) for identification of tumor epithelial cells (clone AE1/AE3 M3515, Dako
Agilent). Slides were mounted using ProLong Diamond Antifade Mountant (Thermo
Fisher, Waltham, MA, USA).
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