Five slides with 3 levels of 3 μm-thick biopsy sections taken 40 μm apart were prepared for each biomarker, yielding a total of 15 levels for each biomarker. To uncover the epitope, heat-mediated antigen retrieval was used: the slides were placed in a preheated Pretreatment Module (Lab Vision Corp., CA) with 100x Citrate Buffer pH 6.0 (DAKO S1699, DAKO Corp., Carpinteria, CA) and steamed for 40 minutes. Then, the slides were placed in a DakoCytomation Autostainer Plus System automated immunostainer and immunohistochemically processed using a labeled streptavidin-biotin method (LSAB2 Detection System [DAKO K0675]) and a monoclonal antibody to each biomarker (for APC, Oncogene OP80 at a concentration of 1:50; for β-catenin, BD Pharmingen [formerly Transduction Laboratories 610154], at a concentration of 1:300; for E-cadherin, Zymed 33–4000 at a concentration of 1:50). For each participant, baseline and follow-up biopsy slides were stained in the same batch, and each staining batch included a balance of participants from each treatment group. The slides, which were not counterstained, were coverslipped with a Leica CV5000 Coverslipper (Leica Microsystems, Inc., IL). Positive and negative control slides were included in each slide staining batch.
Autostainer plus system
Manufactured by Agilent Technologies
Sourced in United States
The Autostainer Plus system is an automated immunohistochemistry (IHC) and in situ hybridization (ISH) staining instrument. It is designed to automate the staining process, providing consistent and reproducible results.
Automatically generated - may contain errors
Lab products found in correlation
Lsab2 detection system, by Agilent Technologies (1 mentions)
Pretreatment module, by Lab Vision (1 mentions)
Aperio scanner system, by Leica (1 mentions)
Envision flex, by Agilent Technologies (1 mentions)
Pt link module, by Agilent Technologies (1 mentions)
Cyclin d1, by Cell Signaling Technology (1 mentions)
P akt ser473, by Cell Signaling Technology (1 mentions)
Gsk3β, by Cell Signaling Technology (1 mentions)
Envision flex reagents, by Agilent Technologies (1 mentions)
Balb c mice, by Janvier Labs (1 mentions)
Cd56 123c3, by Cell Signaling Technology (1 mentions)
Ttf 1, by Cell Signaling Technology (1 mentions)
7 protocols using autostainer plus system
1
Immunostaining Protocol for Biopsy Slides
2
Quantitative COMP Immunohistochemistry in Breast Cancer
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Breast cancer tissue was mounted using FLEX system microscope coated slides. Antigen retrieval was performed with Envision Flex high pH kit (Dako) using a PT-link module (Dako). Tissues were stained with 0.47 μg/ml rabbit polyclonal affinity purified anti-COMP in-house antibody previously evaluated for its specificity (1 (link)), utilizing Envision Flex (Dako) reagents in the Autostainer Plus system according to the manufacturer's protocol (Dako). Slides were scanned with Aperio Scanner system (Leica) at 40X and intensity of COMP evaluated in a blinded fashion using scores: 0 for negative staining, 1 for low expression, 2 for moderate expression and 3 for high expression.
3
Immunohistochemistry Protocol for p21 and Ki-67
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IHC was carried out with Dako Autostainer Plus system (Dako, Carpinteria, CA, USA) as described [29 (link)]. Briefly, sections were deparaffinized, rehydrated and subjected to antigen retrieval in citrate buffer (10 mM, pH 6.0) for 5 mins, and then endogenous peroxidase and alkaline phosphatase activity were blocked with Dual Block for 10 mins. The slides were then incubated overnight at 4°C with p21 (1:75 dilutions) and Ki-67 antibodies (1:100 dilutions). After washing, this was followed by incubation with EnVision secondary antibody for 30 mins at room temperature. Signals were detected by adding substrate hydrogen peroxide using diaminobenzidine (DAB) as a chromogen followed by hematoxylin counterstaining. Finally, the staining result was considered higher expression (intensity 2 or 3 and percent category 2 or 3) or lower expression (intensity 0 or 1, or more but percent category 0 or 1). For each section, the total score was calculated by multiplying the scores of intensity and percentage.
4
Melanoma Tissue Characterization Protocol
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With approval and supervision from the institutional review board and informed consents from all patients, we collected forty melanoma tissues from patients accepted treatment in the First Affiliated Hospital of Xi'an Jiaotong University. All patients participated in this study did not treat with any preoperative therapy, including chemotherapy, immunotherapy and radiotherapy, with their diagnoses determined according to the histopathological evidences.Fresh tissues were carefully washed with sterile PBS (phosphate buffered saline) and stored at -80°C immediately after surgery for subsequent RNA extraction. For IHC assay (immunohistochemical assay), the tissues were fixed with paraformaldehyde, embedded with paraffin and cut into sections with the thickness of 5 μm before staining with primary antibody against CD31 or VEGFB using the DAKO Autostainer Plus system. Pathologist double-blindly viewed and analyzed the sections under high power (× 400) field.
5
Immunohistochemical Analysis of Tumor Markers
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Five-micrometer-thick sections of animal tumor tissues were prepared. A DAKO Autostainer Plus system was employed to perform immunohistochemical staining of p-AKT Ser473 (Cell-Signaling Technology, dilution 1:100, #4060), GSK 3β (Cell-Signaling Technology, dilution 1:100, #12456), and cyclin D1 (Cell-Signaling Technology, dilution 1:50, #2978). Scoring of each tissue section was performed in a double-blinded manner. Each section was examined under a microscope in high-power fields of view (× 400).
6
Immunostaining of Mouse Lung Tissue
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Eight week-old BALB/c mice (Janvier, Saint-Berthevin Cedex, France) were challenged with intranasal PBS containing 50 μg of OMV from P. aeruginosa PAO1 (n = 10) or NTHi 3655 (n = 10) or with PBS only (n = 15). At 0, 24, or 48 h following the challenge, mice were sacrificed, and lungs were retrieved in formaldehyde and sliced into 4 μm sections. For immunohistochemistry staining and counterstaining with hematoxylin, primary anti-mouse vitronectin antibodies (ab62769; Abcam) and EnVision FLEX reagents in an Autostainer Plus system were used according to the manufacturer’s protocol (Dako). Antigen retrieval was performed in a pressure cooker in the Target Retrieval Solution, Citrate pH 6 (Dako).
7
Immunohistochemical Staining of CD56 and TTF-1
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The tissue staining was fixed in 4% paraformaldehyde and embedded in paraffin. Three micrometer thick sections were prepared, and immunohistochemical staining of CD56 (123C3, dilution 1:200, Cell Signaling Technology, Danvers, MA), TTF-1 (dilution D2E8,1:150, Cell Signaling Technology, Danvers, MA) were performed using a DAKO Autostainer Plus system. Each section was examined under a ×200 power field in a double-blinded manner.
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