A2780, A2780pacR or A2780olapR cells for immunohistochemical analysis were grown in 10 cm2 dishes, harvested by scraping, centrifuged (1000 r.p.m., 5 min, RT) and re-suspended in 50 μl Normal Pooled Plasma (CCN-10, Alpha Laboratories, Eastleigh, UK). Suspended cells were pelleted by adding 50 μl Bovine Thrombin (BTUD293, Diagen Reagents Ltd., Thame, UK), and cell pellets immersed in 10% neutral buffered formalin prior to overnight processing on a Leica Peloris II processor using an 8-h xylene standard protocol. Processed cells were then embedded into paraffin wax. Sections (4 μM ) were cut onto Superfrost plus slides (Thermo Scientific)) and dried for 1 h at 60 °C. Antigen retrieval and de-paraffinisation was performed using DAKO EnVision FLEX Target Retreval solution (high pH) buffer in a DAKO PT Link (Dako, Ely, UK). Immunostaining using DAKO EnVision FLEX system on a DAKO Autostainer Link48 was carried out according to the manufacturer's protocol. Sections were incubated with 1/2000 dilution of ABCB1 primary antibody (SC-55510, Santa Cruz Biotechnology) for 30 min. DAKO substrate working solution was used as a chromogenic agent for 2 × 5 min, and sections were counterstained in EnVision FLEX hematoxylin. Negative controls were prepared by replacing the primary antibody with DAKO antibody diluent.
Dako autostainer link 48
Manufactured by Agilent Technologies
Sourced in United States, Denmark, United Kingdom
The Dako Autostainer Link 48 is a compact and automated immunohistochemistry (IHC) and in situ hybridization (ISH) staining instrument. It is designed to handle up to 48 slides in a single run, with the ability to perform multiple staining protocols simultaneously. The instrument provides consistent and reproducible staining results through its precise liquid handling and temperature control capabilities.
Automatically generated - may contain errors
Lab products found in correlation
Pt link, by Agilent Technologies (11 mentions)
Envision flex peroxidase blocking reagent, by Agilent Technologies (7 mentions)
Envision flex target retrieval solution, by Agilent Technologies (6 mentions)
Envision flex, by Agilent Technologies (5 mentions)
Dako pt link, by Agilent Technologies (4 mentions)
Envision flex mouse linker, by Agilent Technologies (4 mentions)
Envision flex hrp, by Agilent Technologies (4 mentions)
Envision flex kit, by Agilent Technologies (4 mentions)
Envision flex hematoxylin, by Agilent Technologies (3 mentions)
Dako mounting medium, by Agilent Technologies (3 mentions)
Dp73 camera, by Olympus (3 mentions)
Target retrieval solution, by Agilent Technologies (2 mentions)
Clone mib 1, by Agilent Technologies (2 mentions)
Clone 8g7g3 1, by Agilent Technologies (2 mentions)
Clone dak p63, by Agilent Technologies (2 mentions)
Envision flex substrate buffer, by Agilent Technologies (2 mentions)
Meyer s hematoxylin, by Merck Group (2 mentions)
Envision flex dab, by Agilent Technologies (2 mentions)
Envision target retrieval solution, by Agilent Technologies (2 mentions)
Envision flex antibody diluent, by Agilent Technologies (2 mentions)
81 protocols using dako autostainer link 48
1
Immunohistochemical Analysis of Drug-Resistant Cancer Cells
2
HER3 Immunohistochemical Staining Protocol
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We performed immunohistochemical (IHC) staining for HER3 as previously described [34 (link), 35 (link)]. Briefly, sections of each sample were deparaffinized and antigen retrieval was performed at high pH (PT Link machine, Dako). The sections were then stained with a rabbit monoclonal antibody against HER3/ErbB3 (1:59 dilution; clone D22C5, Cell Signaling Technology Inc., Danvers, MA, USA) using the Dako autostainer Link48 (Dako, CA, USA) and EnVision Flex Mini Kit (Dako), according to the manufacturer’s instructions. Hematoxylin was used as a nuclear counterstain. HER3-high was defined as an IHC score of 3 + or 2 + , and HER3-low/zero was defined as an IHC score of 1 + or 0 in line with the HER2 testing guidelines for gastroesophageal cancer [36 (link)]. The H-score (range, 0–300) was calculated using the following formula: 3X + 2Y + Z, where X, Y, and Z are the percentages of tumor cells showing strong, moderate, and weak staining intensities, respectively [37 (link)] (Additional file 1 : Table S1A–D). The specificity of this HER3 IHC staining method was validated by confirming no signal detected with an isotype control antibody. Both positive and negative cell line control slides were also incorporated into HER3 staining to verify the specificity at every batch of staining.
3
Immunohistochemistry Profiling of Tissue Microarrays
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TMA blocks were cut into 4-µm sections. IHC reactions were performed using the Dako Autostainer Link48 (Dako, Glostrup, Denmark). Deparaffinisation, rehydration, and epitope retrieval (97°C, 20 min) were performed using a low pH Target Retrieval Solution (Dako/Agilent Technologies, Santa Clara, CA, USA) in a PT- Link (Dako). Subsequently, the sections were washed in Tris-buffered saline and incubated with primary antibodies at room temperature for 20 min. The following specific primary antibodies were used: polyclonal rabbit anti-Periostin (dilution 1:200; code no.NBP1-82472; Novus Biologicals, Littleton, CO, USA), monoclonal mouse anti-Ki-67 antibody (ready-to-use, Clone MIB-1, code IS626; Dako), anti-TTF-1 (ready-to-use, Clone 8G7G3/1, code IR056; Dako), anti-p63 (ready-to-use, Clone DAK-p63, code IR662; Dako), anti-Podoplanin (ready-to-use, clone D2-40 (PDPN), code ISO072; Dako), anti-Vimentin (ready-to-use, clone V9, code GA630; Dako), and anti-αSMA (ready-to-use, clone IS611, code 1A4; Dako). The sections were then visualized using an EnVision FLEX kit (Dako). All slides were counterstained with haematoxylin (Dako). Negative control sections were generated in the absence of the primary antibody.
4
IHC Staining for Ki67 and VEGF
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IHC staining was performed on 4-µm sections using the DAKO Autostainer Link 48 (Dako; Agilent Technologies, Inc.). The tissue epitopes were repaired using the automated water bath heating process in Dako PT Link (Dako; Agilent Technologies, Inc.); the sections were incubated in Tris-EDTA retrieval solution (10 mM Tris, 1 mM EDTA; pH 9.0) at 98°C for 20 min. The sections were subsequently incubated for 50 min at room temperature with the primary anti-Ki67 (cat. no. MIB1; Dako; Agilent Technologies, Inc.) and anti-VEGF (cat. no. VG1; Dako; Agilent Technologies, Inc.) antibodies, diluted at 1:200 and 1:100, respectively, in Dako Envision™ Flex antibody diluents, followed by anti-rabbit immuno-peroxidase polymer (Envision FLEX/HRP) for 20 min at room temperature according to the manufacturer's instructions, and developed with freshly prepared 0.05% 3,3′-diaminobenzidine tetrahydrochloride. Finally, the slides were counterstained with hematoxylin, dehydrated, and mounted.
5
Immunohistochemical Detection of H. Pylori
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Dewaxed paraffin sections were placed in a microwave (10 min, 600 watts) to recover antigens before staining. The tissue samples were probed with rabbit anti-H. Pylori antibodies (B0471, DAKO, Glostrup, Denmark; 1 : 200) using Dako Autostainer Link 48(DAKO). Peroxidase-conjugated streptavidin was used with 3,3-diaminobenzidine tetrahydrochloride for antibody detection, and hematoxylin was used for nuclear counterstaining. Stained slides were analyzed for the presence of the bacteria by an experienced pathologist.
6
Immunohistochemical Detection of AMH
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Slides from TMAs (4 μm-thick) were used for immunohistochemistry (IHC) reactions, which were performed using DakoAutostainer Link48 (Dako, Glostrup, Denmark). In order to deparaffinize, rehydrate and unmask the antigens the sections were boiled in EnVision FLEX Target Retrieval Solution (pH 9, 20 min, 97 °C; Dako) using the PTLink platform (Dako, Glostrup, Denmark). Afterwards, slides were incubated for 5 min with Envision Flex Peroxidase-Blocking Reagent (Dako, Glostrup, Denmark) to block endogenous peroxidase. As primary antibodies (20 min, RT), rabbit polyclonal antibodies against AMH (1:100, ab84952, Abcam, Cambridge, UK) were used. Next, slides were incubated with EnVision FLEX/HRP (20 min, RT), and the reaction was visualized (10 min, RT) with freshly prepared 3,3′-diaminobenzidine (DAB). Additionally, slides were counterstained for 5 min with EnVision FLEX Hematoxylin (Dako, Glostrup, Denmark). Finally, slides were dehydrated in ethanol (70%, 96%, absolute) and xylene, then mounted with Dako Mounting Medium (Dako, Glostrup, Denmark). Slides were evaluated using the Olympus BX41 light microscope (Olympus, Japan). Control tissues included the human prostate.
7
PD-L1 Immunohistochemistry in FFPE Tumors
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PD‐L1 IHC was carried out on 4‐μm sections of FFPE tumour tissue samples using Dako PD‐L1 IHC 28‐8 PharmaDx (Agilent). The test was performed using the EnVision FLEX visualization system on the Dako Autostainer Link 48 and Dako PT Link Pretreatment Module (Agilent). A minimum of 100 viable tumour cells must be present for evaluation. PD‐L1 expression was evaluated only in tumour cells. Scoring was determined according to the tumour proportion score (TPS), which is defined as the percentage of positive viable tumour cells among all viable tumour cells evaluated. A tumour cell was defined as positive for PD‐L1 staining whenever any partial or complete membranous staining was detected. The percentage of PD‐L1‐positive tumour cells was assessed as previously described [20 (link)]. Slides were assessed independently by two pathologists.
8
Tumor Immune Profiling by IHC Staining
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A pathologist (JD) reviewed all of the archival material that was collected within a year prior to the initiation of BCG therapy. The pathologist then selected a single formalin-fixed paraffin-embedded tumor tissue block with the highest tumor grade and stage for each patient. Four-micrometer-thick tissue sections were used for IHC staining (Figure 3 ). We used a CD8 cytotoxic T cell marker (clone C8/144B, Dako, Glostrup, Denmark; dilution 1:100), B cell marker CD20 (clone L26, Dako, Glostrup, Denmark; dilution 1:500), M1 macrophage marker CD11c (clone 5D11, Leica biosystems, Deer Park, NY, USA; dilution 1:100), M2 macrophage marker CD163 (clone MRQ-26, Rocklin, CA, USA; dilution 1:50), and ICOS marker (clone D1K2T, Cell Signaling Technology, Danvers, MA, USA; dilution 1:500) for the identification of specific subpopulations of T cells. CD8, CD11c, and CD163 IHC were performed using Ventana Benchmark Ultra autostainer (Roche Diagnostics, Mannheim, Germany) and CD20 and ICOS using Dako Autostainer Link 48+ (Dako, Glostrup, Denmark). All slides were digitized at 20× magnification (0.5 µm per pixel) using an Aperio® AT2 DX scanner (Leica Aperio Technologies, Vista, CA, USA).
9
Automated Immunohistochemical Staining Protocol
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All four immunohistochemical analyses were carried out on a DAKO Autostainer Link 48 (DAKO, Ely, England, UK) using DAKO pre-programmed protocols, available with the Autostainer. Antigen retrieval was performed in the accompanying PT-Link chamber with High pH DAKO Target Retrieval Solution, according to manufacturer's instructions (DAKO, Ely, England, UK). Slides were rinsed with DAKO wash buffer prior to loading into the Autostainer. DAKO ready-to-use antibodies were used for MLH1 (IR079), MSH2 (IR085) and MSH6 (IR086). DAKO PMS2 (M3674) was used at a dilution of 1:40. Sections from the two validation TMAs were stained with each Ab, then corresponding whole sections were also stained in cases where the cores appeared negative or equivocal or for cases where all cores had been lost from the TMA section. Tumours were deemed positive, if any proportion of the tumour nuclei was positively stained, or negative, where all discernible tumour nuclei were negative, in the local presence of positively staining stromal and infiltrating lymphocytic cells (figure 2 ). Any samples appearing wholly negative with respect to both tumour and stromal components were deemed to be of indeterminate status.
10
Immunohistochemical Staining of ZYX Protein
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IHC reactions were performed on 4 µm thick TMA sections placed on Superfrost Plus slides (Menzel Gläser, Braunschweig, Germany). The reactions were performed using the EnVision Flex System (Dako, Glostrup, Denmark). Deparaffinization, rehydration, and antigen retrieval (97 °C, 20 min) were performed in low-pH EnVision FLEX Target Antigen Retrieval Solution (pH = 6) using the PT Link (Dako, Glostrup, Denmark). Dako Autostainer Link48 was used for performing IHC reactions (Dako, Glostrup, Denmark). Endogenous peroxidase activity was blocked by a 5 min incubation with EnVision FLEX Peroxidase-Blocking Reagent (Dako, Glostrup, Denmark). The sections were incubated for 20 min with anti-ZYX monoclonal antibody (1:50, 2D1 clone, catalogue no. sc-293448, Santa Cruz Biotechnology, Dallas, TX, USA) followed by EnVision FLEX + MOUSE LINKER for 15 min. Next, the sections were incubated for 20 min with EnVision FLEX/HRP secondary antibody (Dako, Glostrup, Denmark). DAB+ Chromogen (Dako, Glostrup, Denmark) was used to visualize the reaction. Hematoxylin was used to visualize cell nuclei according to the manufacturer’s instructions (Dako, Glostrup, Denmark).
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