Formalin fixed, paraffin embedded (FFPE) tissue sections (5 µm thickness) from heart and skeletal muscle tissues selected from fish with HSMI, were mounted using Superfrost plus (Thermo Fisher Scientific) slides. Sections were baked at 60°C for 2 hrs in HybEZ™ II oven (Advanced Cell Diagnostics, catalog #321720) prior to deparaffinization with absolute ethanol (100%) and fresh xylene. Initial blocking was done with hydrogen peroxide (Advanced Cell Diagnostics) for 10 min at room temperature (RT). RNAscope antigen retrieval reagent (Advanced Cell Diagnostics, catalog #322000) was used for 15 min at 99°C and slides were further incubated with RNAscope protease plus reagent for 15 min at 40°C in the HybEZ™ II oven following manufacturer guidelines. Immedge hydrophobic barrier pen (Vector Laboratories, Burlingame, CA) was used to make hydrophobic barrier around tissue areas over slides for further probe hybridization procedures.
Hybez 2
Manufactured by Advanced Cell Diagnostics
Sourced in United States
The HybEZ™ II is a laboratory equipment designed for in situ hybridization (ISH) experiments. It provides a controlled environment for efficient and consistent hybridization of nucleic acid probes to target sequences in tissue samples.
Automatically generated - may contain errors
Lab products found in correlation
Hydrogen peroxide, by Advanced Cell Diagnostics (1 mentions)
Immedge hydrophobic barrier pen, by Vector Laboratories (1 mentions)
Rnascope antigen retrieval reagent, by Advanced Cell Diagnostics (1 mentions)
Rnascope protease plus reagent, by Advanced Cell Diagnostics (1 mentions)
Superfrost plus, by Thermo Fisher Scientific (1 mentions)
Hybez 2 oven, by Advanced Cell Diagnostics (1 mentions)
Alkaline phosphatase conjugated sheep anti dig antibody, by Roche (1 mentions)
2 protocols using hybez 2
1
In Situ mRNA Detection in FFPE Fish Tissues
2
CCNE1 In Situ Hybridization Protocol
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A previously published in‐house CISH protocol using a commercial digoxigenin (DIG)‐labeled CCNE1 DNA probe (Empire Genomics, Buffalo, NY, USA) was used.15 Deparaffinized 4‐μm tissue microarray sections were pretreated with proteinase K (3 min) and citrate‐based antigen retrieval buffer at 80°C (1 h) followed by pepsin (45 sec), and then dehydrated and air‐dried. Hybridization with the DIG‐labeled CCNE1 probe was performed at 37°C for 16 to 18 hours in HybEZ II (Advanced Cell Diagnostics, Minneapolis, MN, USA). A levamisole solution was used (15 min) to remove endogenous alkaline phosphatase activity, followed by a blocking solution (30 min) of 10% normal sheep serum, 2% bovine serum albumin, and 0.05% Tween‐20. An alkaline phosphatase‐conjugated sheep anti‐DIG antibody (dilution 1:800; Roche, Basel, Switzerland) was incubated for 2 h. An alkaline phosphatase substrate was applied, and the reaction was stopped with 50 mM Tris, 150 mM NaCl, and 10 mM KCl buffer when slides reached the desired intensity of staining. Counterstaining was performed with hematoxylin.
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