Formalin-fixed, paraffin-embedded tissues were cut at a thickness of 4 to 5 μm. Dual- and single-marker staining were performed on sections according to a standard immunostaining protocol using a Ventana Benchmark XT Automated Stainer, following the manufacturer’s instructions. Briefly, the sections were heated for 1 hour at 58°C, deparaffinized, and dehydrated in a graded alcohol series. Antigen retrieval was performed using Cell Conditioning 1 (Ventana) Tris/Borate/EDTA for 60 minutes with a Ventana Benchmark XT Automated Stainer followed by dual-marker immunostaining for p40/napsin A and CK5/6/TTF1, according to the manufacturer’s instructions. The sections were blocked with hydrogen peroxide and normal goat serum and then incubated with a mixture of anti-p40 and anti-napsin A or anti-CK5/6 and anti-TTF1 antibodies at ratios of 1:1 for 30 minutes at 37°C. The antibody concentrations were about 1.0 µg/mL, respectively. The color was developed with 3,3’-diaminobenzidine (DAB), and the sections were then counterstained with hematoxylin. Positive controls were single-stained for p40, napsin A, CK5/6, or TTF-1. Single-marker immunostaining for p40, napsin A, CK5/6, and TTF1 was also performed synchronously using an UltraView DAB Detection Kit and Ventana Benchmark XT Automated Stainer, according to the manufacturer’s instructions.
Cell conditioning 1
Manufactured by Roche
Sourced in United States
Cell Conditioning 1 is a laboratory instrument designed for the preparation and processing of biological samples. It provides controlled environmental conditions to maintain the integrity of cells during various experimental procedures.
Automatically generated - may contain errors
Lab products found in correlation
Ez prep solution, by Roche (11 mentions)
Ventana discovery xt, by Roche (10 mentions)
Benchmark xt, by Roche (10 mentions)
Dako antibody diluent, by Agilent Technologies (8 mentions)
Ultraview universal dab detection kit, by Roche (7 mentions)
Ez prep, by Roche (5 mentions)
Ultraview dab detection kit, by Roche (4 mentions)
Hematoxylin and bluing reagent, by Roche (4 mentions)
Vs 110 microscope, by Olympus (3 mentions)
Benchmark ultra, by Roche (3 mentions)
Alx 804 717, by Enzo Life Sciences (3 mentions)
Ventana benchmark xt system, by Roche (3 mentions)
Iview dab detection kit, by Roche (2 mentions)
Discovery xt, by Roche (2 mentions)
Benchmark ultra autostainer, by Roche (2 mentions)
Olyvia software, by Olympus (2 mentions)
Mouse monoclonal antibody to human nampt, by Enzo Life Sciences (2 mentions)
Ultraview universal dab detection system, by Roche (2 mentions)
Chromomap kit, by Roche (2 mentions)
Ribo cc, by Roche (2 mentions)
42 protocols using cell conditioning 1
1
Dual-Marker Immunostaining for Lung Cancer
2
PTEN Expression Analysis in Tissues
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Immunohistochemical analysis of PTEN expression (1:100 dilution, Cell Signaling Technologies) was performed on 3-μm paraffin sections of formalin-fixed, paraffin-embedded tissues using the Ventana Discovery XT automated stainer (Ventana Medical Systems, Tucson, AZ, USA). After deparaffinization, antigen retrieval was carried out in CC1 buffer (Cell Conditioning 1; citrate buffer pH 6.0, Ventana Medical Systems). PTEN expression was scored independently by two investigators (Y. H. and N. Y.) based on stain intensity and extent. Immunohistochemical scoring was conducted in a manner entirely blinded to all clinical and biological variables. The intensity of positive staining was scored from 0 to 2 as follows: 0 (none), 1 (weak; intensity < positive control), 2 (strong; intensity ≥ positive control). Positive staining was assigned using a semi-quantitative, five-category grading system: 0, <5% positive cells; 1, 6–25% positive cells; 2, 26–50% positive cells; 3, 51–75% positive cells; 4, 76–100% positive cells. Addition of the two values gives the total score, and a score <4 was considered PTEN-negative.
3
Immunohistochemical Staining of Protein Targets
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Immunohistochemical (IHC) staining was performed using the BenchMark ULTRA slide staining system (Ventana Medical Systems, USA). Briefly, TMA tissue sections were deparaffinized and rehydrated with EZprep concentrate (10×) solution (Ventana Medical Systems). Antigen retrieval was performed with cell conditioning 1 (Ventana Medical Systems) at 95°C for 30 min. Slides were then treated with iView DAB (3,3'-diaminobenzidine tetrahydrochloride) Inhibitor (Ventana Medical Systems) and incubated with 100 µL primary antibody (1∶100) for 90 min. The following primary antibodies were used for IHC staining: pAMPK (Thr 172), pACC (Ser 79), and phosphorylated extracellular signal-regulated kinase (pErk) (Thr 202/Tyr 204) (All Cell Signaling Technology; Danvers, MA, USA) and immunoglobulin G (clone SP3) (Thermo Fisher Scientific, USA). These antibodies were chosen because they have been investigated in other studies [15] (link)–[17] (link). Bound antibody was detected using the iView DAB Detection Kit (Ventana Medical Systems) according to the manufacturer’s instructions.
4
ALK IHC Protocol for NSCLC
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The presence of ALK overexpression was assessed by immunohistochemistry (IHC) staining using 4-μm-thick sections. Ganglion cells present in sections of the appendix were used as positive controls, and in negative controls, the primary antibody was omitted. Immunohistochemical reactions were performed at Ventana Benchmark XT using the Ventana ALK (D5F3) CDx assay (Ventana, Tucson, AZ, clone 790–4796) according to the manufacturer. In brief, slides of the NSCLC tumor were subjected to deparaffinization using EZ Prep (Ventana, Tucson, AZ) and antigen retrieval was performed using Cell Conditioning 1 (Ventana, Tucson, AZ). Tissue sections were then incubated with anti-ALK antibody (clone D5F3, Ventana, Tucson, AZ) for 20 min. The OptiView DAB IHC Detection Kit (Ventana, Tucson, AZ) and OptiView Amplification Kit (Ventana, Tucson, AZ) were used according to the manufacturer’s recommendations for the visualization of the bound primary antibody. The ALK stain was considered positive if at least one cell presented strong dark brown cytoplasmic staining as stated in the kit’s manual as previously described [20 (link)].
5
Immunohistochemical Detection of PPARγ
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Lung tissues were fixed, embedded, sectioned at a thickness of 4 µm, and placed on slides for labeling using the Discovery XT automated immunohistochemistry staining system (Ventana Medical Systems, Inc., Tucson, AZ, USA). Tissue sections were deparaffinized and rehydrated. For antigen retrieval, a Cell Conditioning 1 (Ventana Medical Systems) standard (pH 8.4 buffer containing tris/borate/EDTA) was used. The sections were incubated with the anti-PPARγ antibody at 37 °C for 32 min, washed, and incubated with a secondary antibody for 20 min. After successive washes, the slides were incubated with 3,3-diaminobenzidine H2O2 substrate at 37 °C for 8 min, followed by counterstaining with hematoxylin and a bluing reagent. Stained cells were observed under a microscope (EVOS XL Core Cell Imaging System, Thermo Fisher Scientific).
6
Immunohistochemical Analysis of NF-κB Activation
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Four-micrometre-thick sections of each TMA block were subjected to antigen retrieval in Cell Conditioning 1 (#950–124, Ventana Medical Systems, Tucson, AZ, US) for 90 minutes and then stained using the BenchMark XT autostainer (Ventana Medical Systems). Pre-diluted anti-NF-kB p65 mouse monoclonal antibody (sc-8008 [F-6], Santa Cruz Biotechnology, Santa Cruz, CA, US) at 1:100 was manually added to the slides and incubated at 37°C for 60 minutes. Antibody binding was revealed using the ultraView Universal DAB Detection Kit (#760–500, Ventana Medical Systems). Counterstaining was achieved using hematoxylin and bluing reagents (#760–2021 and #760–2037, Ventana Medical Systems). Tissues were dehydrated and mounted using Sub-X Mounting Medium (Leica Microsystems, Concord, ON, Canada). All sections were scanned using a VS-110 microscope with a 20× 0.75 NA objective and a resolution of 0.3225 μm (Olympus Canada, Richmond Hill, ON, Canada).
7
Quantifying CD8+ T Cells in FFPE Samples
8
Immunohistochemistry of c-Met in Formalin-Fixed Paraffin-Embedded Tumors
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After ex vivo imaging, the specimen was formalin-fixed, sectioned and then paraffin-embedded. IHC of the formalin-fixed paraffin-embedded tumor samples was performed on a BenchMark Ultra autostainer (Ventana Medical Systems, Oro Valley, AZ, USA). In brief, 3 µm paraffin sections were deparaffinized with the EZ prep solution (Ventana Medical Systems), and heat-induced antigen retrieval was carried out using Cell Conditioning 1 (Ventana Medical Systems). Hereafter, sections were incubated with an anti-c-MET antibody (clone SP44; Roche Diagnostics, Rotkreuz, Switserland), and an UltraView Universal DAB Detection Kit (Ventana Medical Systems) was used to visualize the c-Met expression. Slides were counterstained with Hematoxylin and Bluing Reagent (Ventana Medical Systems). Due to the lack of a quantitively read-out in routine immunohistochemistry, the c-Met expression levels were visually scored by a dedicated pathologist. Scoring was based on the intensity of the UltraView signal (four classifications were used: no staining, weak, moderate and strong, respectively −, +, ++, +++). Both membrane and cytoplasmic staining were noted. Since c-Met is considered a membrane-bound biomarker [9 (link)], membranous staining was leading in the scoring.
9
Immunohistochemistry Protocol for FFPE Sections
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The antibodies used for immunohistochemistry in this study are shown in Table 1 . Briefly, FFPE blocks were sectioned at a thickness of 3 um and then deparaffinized and rehydrated using xylene and alcohol solutions, respectively. Sections were then stained using the VentanaDiscoversy XT automated stainer (Ventana Medical System, Tucson, AZ, USA). Antigen retrieval was achieved by soaking sections in a CC1-buffered solution (Cell Conditioning 1; citrate buffer Ph 6.0, Ventana Medical System). The appropriate positive and negative controls were included together with the study sample for staining.
10
Immunostaining of Tissue Microarray Samples
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Immunostaining was performed on 4 µm TMA sections (Discovery and COEUR) using the Benchmark XT autostainer (Ventana Medical Systems, Roche, Rotkreuz, Switzerland). Antibody staining conditions were based on the manufacturer’s datasheet for each marker. Antigen retrieval was performed using Cell Conditioning 1 (Tris-EDTA buffer, pH 7.8, for KRT7, KRT18, KRT19, and VIM) or 2 (citrate buffer, pH 6.0, for E-CADH) (Ventana Medical Systems) and slides were then incubated for one hour with the primary mouse monoclonal antibodies against E-CADH (1/100, G10 sc-8426, Santa Cruz Biotechnology, Dallas, TX, USA), KRT7 (1/200, CLONE OV-TL 12/30, Thermo Scientific, Waltham, MA, USA), KRT18 (1/200, sc-6259, Santa Cruz Biotechnology), KRT19 (1/200, MS-198-P, Thermo Scientific) or VIM (1/200, sc6260, Santa Cruz Biotechnology). Epithelial cells were identified using a highly sensitive cocktail of rabbit monoclonal antibodies against KRT8/18 (FLEX, clone EP17/EP30, Dako, Mississauga, ON, Canada). Slides were incubated with secondary fluorescent antibodies for 45 min and cell nuclei were stained with DAPI. Slides were mounted with coverslips using Fluoromount medium (F4680, Millipore-Sigma, Oakville, ON, Canada).
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