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.
Lsab2 detection system
Manufactured by Agilent Technologies
The LSAB2 Detection System is a laboratory equipment designed for the detection and analysis of target analytes. It utilizes a specialized technique to provide reliable and accurate results. The system's core function is to facilitate the identification and quantification of specific substances within a sample.
Automatically generated - may contain errors
Lab products found in correlation
Autostainer plus system, by Agilent Technologies (1 mentions)
Pretreatment module, by Lab Vision (1 mentions)
Superfrost slide, by Thermo Fisher Scientific (1 mentions)
Dab substrate kit, by Abcam (1 mentions)
Peroxidase blocking reagent, by Agilent Technologies (1 mentions)
Mouse igg1, by Agilent Technologies (1 mentions)
Depex mounting media, by Electron Microscopy Sciences (1 mentions)
2 protocols using lsab2 detection system
1
Immunostaining Protocol for Biopsy Slides
2
Immunohistochemical Analysis of Prostate Tissues
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Tissues were fixed in 4% PFA for 24 h, dehydrated with an ascending ethanol series followed by xylene, and paraffin embedded. Tissues were sectioned at 4–6 µm and collected onto SuperFrost slides (ThermoFisher Scientific). Sections were stained with haematoxylin and eosin (H&E), and with anti-PSA (Ventana Cat #: 760-2506) and anti-Ku70 (ab58150) antibodies. For antibody staining, sections were treated with peroxidase blocking reagent (Dako, S2001) for 5 min at room temperature, antigen-retrieved (10 mM sodium citrate, pH 6.0) at high temperature/pressure for 1 min, and blocked with Protein Block Serum Free Medium (Dako, X0909) for 30 min at room temperature. Primary antibodies (anti-PSA at 1:100, and anti-Ku70 at 1:6000) in 0.5% BSA were incubated on the slides overnight at 4 °C. The DAKO LSAB2 detection system (Cat #K0675) was used per manufacturer’s instruction to secondarily label the primaries. A DAB substrate kit (abcam, K3466) was used to develop the chromogenic signal. Sections were counterstained with Mayer’s hematoxylin, followed by cover-slipping in DePex mounting media (Electron Microscopy Sciences, Fort Washington, PA, USA). Negative controls were prepared by substituting primary antibody with mouse IgG1 (Dako, X0931).
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