Serial sections of H&E staining were selected for double immunohistochemical staining. The distinguishing abilities of double immunohistochemical staining using D2-40 combined with factor-VIII were studied. The BenchMark® automated slide-processing system (Roche Diagnostics, Tokyo, Japan) was used according to the manufacturer's protocol. The deparaffinization step was performed for 16 min at 75°C using EZ Prep (Roche Diagnostics). The heat treatment step was performed for 60 min at 100°C using Cell Conditioning 1 solution (Roche Diagnostics). The slides were incubated with D2-40 antibody (cat. no. M3619, clone D2-40, monoclonal mouse; dilution, 1:6; Dako; Agilent Technologies, Inc., Santa Clara, CA, USA) for 32 min at 42°C. The D2-40 protein was visualized using the iVIEWTM 3,3′-Diaminobenzidine Detection Kit (Roche Diagnostics). The same slides were incubated with factor-VIII antibody (cat. no. 518101206, Factor-VIII-related antigen®, polyclonal rabbit antibody; dilution, 1:4; Roche Diagnostics) for 32 min at 42°C subsequent to 100°C heat treatment using Cell Conditioning 1 solution (Roche Diagnostics) for 8 min. Factor-VIII protein was visualized using the ultraVIEWTM Universal Alkaline Phosphatase Red Detection Kit (Roche Diagnostics) followed by counterstaining with Hematoxylin II (Roche Diagnostics) and Bluing Reagent (Roche Diagnostics).
Ez prep
Manufactured by Roche
Sourced in United States
The EZ Prep is a lab equipment product designed for sample preparation. It provides a convenient and efficient way to process samples prior to analysis. The core function of the EZ Prep is to prepare samples for downstream applications.
Automatically generated - may contain errors
Lab products found in correlation
Benchmark xt, by Roche (10 mentions)
Discovery xt, by Roche (10 mentions)
Optiview dab ihc detection kit, by Roche (7 mentions)
Cell conditioning 1, by Roche (5 mentions)
Ab15580, by Abcam (5 mentions)
Ventana benchmark xt, by Roche (5 mentions)
Cc1 solution, by Roche (4 mentions)
Reaction buffer, by Roche (4 mentions)
Chromomap dab detection kit, by Roche (4 mentions)
Bluing reagent, by Roche (3 mentions)
Hematoxylin 2, by Roche (3 mentions)
Benchmark ultra, by Roche (3 mentions)
Ultraview universal dab detection system, by Roche (3 mentions)
Cell conditioning 1 cc1, by Roche (3 mentions)
Optiview dab detection kit, by Roche (3 mentions)
Ventana benchmark ultra, by Roche (2 mentions)
Iview dab detection kit, by Roche (2 mentions)
Hematoxylin 1, by Roche (2 mentions)
Ab52625, by Abcam (2 mentions)
Ab5694, by Abcam (2 mentions)
47 protocols using ez prep
1
Dual Immunohistochemistry for D2-40 and Factor-VIII
2
Immunohistochemical Staining of FFPE Tissue Sections
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Four-μm FFPE tissue sections were stained by immunohistochemistry on the Ventana BenchMark Ultra automated slide stainer (Ventana Medical Systems, Roche, Oro Valley, AZ) using standard methods. In brief, sections were deparaffinized using the detergent (EZ-prep, Ventana, Roche, Oro Valley, AZ), heat (72°C) and vortex mixing. Endogenous peroxidase activity was blocked by incubation with 3.0% hydrogen peroxide solution, contained in the OptiView DAB IHC Detection Kit (Ventana, catalog no. 760–700). Heat induced epitope retrieval was applied in order to unmask epitopes, crosslinked as a result of the formalin fixation by heating to 100°C for 32 minutes in acidic buffer. Primary polyclonal rabbit anti-human antibodies against aldolase A (anti-ALDOA) and GAPDH (Product nos. HPA004177 and HPA040067, Atlas Antibodies, Stockholm, Sweden) were diluted (1:200 and 1:1000, respectively) in a Tris buffered diluent with a pH of 7.2, 15 mmol/L NaN3 and protein (Dako catalog no. S202230-2) and incubated for 32 minutes at 36°C. Primary antibody binding was detected with the OptiView DAB IHC Detection Kit (Ventana, catalog no. 760–700) which specifically detects rabbit primary antibodies bound to antigens in the tissue sections. Sections of appendix, tonsil, liver and pancreas were included on each slide as positive/negative controls [23 (link)].
3
NFAT5 Immunohistochemical Quantification
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Immunostaining was performed on formalin fixed, paraffin-embedded archival tissue. The paraffin blocks were sliced into 2.5 μm thick sections onto glass slides. The slides were loaded onto the automated slide stainer on the VENTANA BenchMark Series Instruments (Roche Diagnostics, Mannheim, Germany) for staining. The paraffin sections were deparaffinized with Ez prep (#950-102, Roche Diagnostics). Antigen retrieval was achieved by incubating slides with CC1 (#950124, Roche Diagnostics) for 64 min at 37 °C. Endogenous peroxidases were quenched by incubating the slides with pre-primary peroxidase inhibitors. The slides were then incubated with a primary antibody for NFAT5 (#NB120-3446, Novus Bio, Wiesbaden-Nordenstadt, Germany, 1:250) for 20 min at 37 °C. The bound primary antibody was detected using an OptiView DAB IHC Detection Kit (#760-700, Roche Diagnostics) following the manufacture’s protocol. The immunohistochemical reaction was assessed with Nikon Eclipse E200 light microscope with Nikon DS-Fi1 digital camera (Nikon, Amstelveen, The Netherlands). Complete negative staining equals a score of 0, 0–10% score 1, 10–50% score 2, and >50% score 3. This analysis was independently performed by 2 pathologists (A.S., I.P.).
4
Immunohistochemical Staining of Liver Tissue
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Immunohistochemical staining of sections from the liver tissue samples was performed using a Ventana BenchMark GX (Roche Diagnostics, Basel, Switzerland). In brief, after deparaffinisation using EZ-Prep (Roche Diagnostics) and antigen retrieval using Cell Conditioning 1 buffer at 95°C for 32 min, sections were incubated with primary antibodies. The primary antibodies were anti-survivin (cat. no. ABP54792, polyclonal, dilution 1 : 200; R&D systems, California, USA). Immunoreactivity was considered based on nuclear staining and by counting 100 hepatocytes under a high-power field. The severity of immunoreactivity was assigned by percentage as positive cell number: Grade 0: no staining (Figure 1 ), Grade 1: 1–20% (Figure 2 ) and Grade 2: > 20% staining (Figure 3 ).
5
Immunohistochemical Analysis of Mouse Tumors
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Immunohistochemistry analysis of mouse tumors was performed as described previously4 (link)5 (link)27 (link). Briefly, formalin fixed, paraffin embedded 4 μm sections of the xenografts were dewaxed with Ventana EZ Prep and endogenous peroxidase activity was blocked using the Ventana’s Universal DAB inhibitor. Primary antibodies against Oct4, PLEC, VIM and TUBB2A were diluted according to the instruction provided by the manufacturer and sections were stained using a Ventana Benchmark Immunostainer (Ventana Medical Systems, Inc, Arizona, USA). Detection was performed using Ventana’s Ultra View DAB detection kit (Roche/Ventana, Arizona, USA) using the method described previously4 (link). Tumor sections were counter stained with Ventana Haematoxylin and Blueing Solution. Immunohistochemistry images were captured and analysed by using Aperio ImageScope v12.1.0.5029 as described previously27 (link).
6
Immunofluorescence and Immunohistochemical Staining
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For immunofluorescence, cells were fixed with 4% paraformaldehyde in PBS for 15 min at room temperature. The cells were washed three times with PBS for 5 min, then permeabilized with PBS containing 0.2% Triton X-100 and 0.5% normal goat serum (GIBCO) for 5 min on ice. The cells were incubated at room temperature with PBS with 0.5% normal goat serum (GIBCO) three times for 5 min, then with the primary antibodies for 60 min, followed by the Cy3- or Alexa488-conjugated secondary antibodies for 60 min. The cells were washed with PBS three times for 10 min each. DNA was counterstained with 1 μg ml−1 DAPI.
For immunohistochemical staining, sections in a tissue microarray (Biomax, BR1504) were processed with automated IHC staining system, BenchMark XT (Ventana). The sections were de-paraffinized in EZ prep (Ventana) at 72 °C for 4 min and then incubated in Immunoblock (DS Pharma Biomedicals) at 37 °C for 12 min to block endogenous peroxidase. Antigens were retrieved by incubating 95 °C for 8 min, then treated with rabbit monoclonal anti-human ERα clone SP1 (Ventana, 790–4325, used without dilution) at 37 °C for 36 min. The antigens were visualized by avidin–biotin-peroxidase complex method using iVIEW DAB detection kit (Ventana). The slides were then counterstained with haematoxylin at 37 °C for 8 min.
For immunohistochemical staining, sections in a tissue microarray (Biomax, BR1504) were processed with automated IHC staining system, BenchMark XT (Ventana). The sections were de-paraffinized in EZ prep (Ventana) at 72 °C for 4 min and then incubated in Immunoblock (DS Pharma Biomedicals) at 37 °C for 12 min to block endogenous peroxidase. Antigens were retrieved by incubating 95 °C for 8 min, then treated with rabbit monoclonal anti-human ERα clone SP1 (Ventana, 790–4325, used without dilution) at 37 °C for 36 min. The antigens were visualized by avidin–biotin-peroxidase complex method using iVIEW DAB detection kit (Ventana). The slides were then counterstained with haematoxylin at 37 °C for 8 min.
7
ALK Immunohistochemistry Protocol
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ALK IHC was carried out on 3-μm thick tissue using a Ventana automated immunostainer (Ventana Medical Systems, Tucson, AZ) according to the manufacturer's protocol. Briefly, the slides were deparaffinized using EZ Prep (Ventana Medical Systems) at 75°C for 4 minutes and heat pretreatment at 100°C for 20 minutes. The antibody for ALK (mouse monoclonal, clone 5A4, Novocastra, Newcastle, United Kingdom) was diluted to 1:30 and incubated at 42°C for 2 hours. Signals were detected with an iView detection kit (Ventana Medical Systems) based on a streptavidin-biotin method. Mayer's hematoxylin was used for counterstaining for 2 minutes at room temperature. ALK expression was semiquantitatively assessed based on the intensity of staining and the proportion of stained cells and scored by two independent pathologists (IG Do and KM Kim). An IHC score was assigned as 0 (no staining), 1+ (faint cytoplasmic staining in ≤10% of tumor cells), 2+ (moderate, smooth cytoplasmic staining), and 3+ (intense, granular cytoplasmic staining). IHC scores of 2+ or 3+ were regarded as ALK IHC positive.
8
Immunohistochemical Detection of ROS1 Protein
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Protein expression of ROS1 was detected by immunohistochemical staining in the Korean cohort, and it was not performed in the Singapore cohort due to lack of unstained slides. Tissue microarray sections were stained using the Ventana automated immunostainer BenchMark XT (Ventana Medical Systems, Tucson, AZ). The slides were dried at 60°C for 1 hour and deparaffinized using EZ Prep (Ventana Medical Systems) at 75°C for 4 minutes. Cell conditioning was performed using CC1 solution (Ventana Medical Systems) at 100°C for 8 minutes. ROS1 antibody (rabbit monoclonal, clone D4D6, Cell Signaling Technology, Danvers, MA) was diluted to 1:10, followed by treatment, and incubation at 37°C for 2 hours. Signals were detected using the OptiView DAB IHC Detection Kit (Ventana Medical Systems). Counterstaining was performed using Hematoxylin I (Ventana Medical Systems) for 4 minutes at room temperature.
9
Automated Phosphorylated Antibody Detection
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Immunohistochemistry was performed on an automated VENTANA Discovery XT staining instrument according to the manufacturer’s recommendations. Five different phosphorylated antibodies were utilized in this study from Cell Signaling Technologies: Phospho-AKT (clone D9E, dilution 1:50, Ser473, #4060), Phospho-ERK (clone 20G11, dilution 1:400, Thr202/Tyr204, # 4376), Phospho-SRC (clone D49G4, dilution 1:200, Tyr416. #6943), Phospho-STAT3 (clone D3A7, dilution 1:30, Tyr705, #9145), and Phospho-SMAD2 (clone 138D4, dilution 1:80, Ser465/467, #3108). Slides were deparaffinized using EZPrep (Ventana Medical Systems) at 90 °C, antigen retrieval, and antibodies conditions followed package inserts. Slides were developed using OptiView DAB detection kit (Ventana Medical Systems) and counterstained with hematoxylin. Whole slide images were obtained using an Aperio slide scanning system.
10
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)].
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