In brief, sections were deparaffinized (baking at 60 °C) and subject to antigen retrieval in a using the EZ-Retriever System V.2 (BioGenex, S R, California). Samples were blocked to reduce non-specific binding of the antibodies and incubated with specific primary antibody. Primary antibody binding was visualized using fluorophore-conjugated secondary antibody. For chromogenic staining poly-HRP secondary antibody with DAB chromogen were used and tissue sections were counterstained by Hematoxylin. Appropriate controls were put up to validate the experiments. All the antibody reactions were performed in dark at room temperature (25 °C).
Ez retriever system v 2
Manufactured by BioGenex
Sourced in United States
The EZ-Retriever System V.2 is a laboratory instrument designed for automated sample retrieval. It features a robotic arm that can access and retrieve samples from various storage locations within the system.
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Lab products found in correlation
3 protocols using ez retriever system v 2
1
Immunohistochemical Staining Protocol
2
Formalin Fixation and Paraffin Embedding for Microscopy
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We submerged the tissue samples in 10% phosphate-buffered formalin for tissue fixation for 24 hours. We vacuum-embedded the fixed tissues in melted paraffin to obtain tissue blocks. We cut the formalin-fixed paraffin-embedded (FFPE) tissue into thin sections of 5 m and mounted them on glass slides coated with poly-L-lysine (0.1% w/v in water, Cat. No. P8920, Sigma Aldrich, Germany). We then removed the paraffin by incubating slides dipped in warm (60 ∘C) xylene for 30 min, followed by immersion of the slides in 100% ethanol for 5 min. We performed antigen retrieval on the sections using Tris-EDTA buffer (pH 9.0) using EZ-Retriever System V.2 (BioGenex) with the standard microwave protocol. We dehydrated the samples in graded alcohol (50% and 70%, 5 min each) up to 100%, 45 min followed by air-drying them overnight, and 60-min oven-baking at 45 ∘C before microscopy.
3
Immunohistochemical Staining of Tissue Sections
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The tissue sections were deparaffinized and rehydrated for staining with H and E. The sections were dehydrated following staining through upgrades of alcohol and mounted. [ 19 ] Immunohistochemistry Tissue sections were baked and subsequently deparaffinized and hydrated for antigen retrieval in a 10 mM citrate buffer (pH 6.0) using EZ-Retriever System V.2 (BioGenex, San Ramon, California, USA) and immunostained using the kit (i.e., Super Sensitive Polymer-HRP IHC Detection System, Cat. No. QD400-60K, BioGenex). Sections were incubated with primary antibodies (antihuman p63 clone 4A4, Cat. No. AM418-5M; E-cadherin, clone EP700Y, Cat. No. ab40772, Abcam, Cambridge, UK; and anti-CD105 clone 4G11, Cat. No. AM441-5M, BioGenex). p63 and CD105 antibodies were 'ready to use', whereas a dilution of 1:500 was used for E-cadherin. Primary binding of antibody was visualized using a horseradish peroxidase conjugated secondary antibody using the chromogen 3,39-diaminobenzidine and counterstained with hematoxylin. Appropriate controls were put up to validate the experiments.
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