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How to use EZ-Batch Tray

De-paraffinization

De-paraffinization is performed to ensure complete removal of the paraffin from FFPE samples to allow for the probes to penetrate the target RNA after adequate pretreatment.

Endogenous Peroxidase Blocking

RNAscope H2O2 step is performed during the RNAscope assay to block endogenous peroxidase enzyme activity to prevent hazy background after detection.Note: Pretreatment 1 refers to RNAscope H2O2 reagent and is the first pretreatment perfomed on your samples for Chromogenic assays.

Target Retrieval (Boiling)

Target Retrieval step is a heat induced epitope retrieval method that is necessary to reverse the cross-linking caused by the formalin fixation step. Note: Pretreatment 2 refers to Target Retrieval reagent.For an alternative steamer protocol refer to the appendix of User Manual Part 2 Brown and Red.

Protease Plus (Protease Digestion)

Protease Plus is a broad spectrum protease that is intended to permeabilize the samples adequately to allow the probes to reach the target mRNA.Note : Pretreament 3 refers to Protease Plus reagent.

Target Probe Hybridization

ACD provides properietary double "ZZ" oligo probes designed to hybridize to your specific RNA target 

Amplification (Amp1-Amp6) with Wash Step

RNAscope detection reagents amplify hybridization signals via sequential hybridization of amplifiers.

DAB Colormetric Reaction

Chromogenic detection is based on the enzyme substrate reaction which leads to the formation of an insoluble precipitate visualized in the form of punctate dots for the RNAscope assay.

Gill's Hematoxylin Counter Stain

Hematoxylin staining is a counterstain used to provide a contrast to better visualize the signal and to observe  the morphology of the sample and identify the localization of the signal.

Tissue Dehydration

The tissue dehydration step after the counterstaining step results in removal of excess moisture which provides better  tissue morphology and preservation of the signal.

Mounting

The final step in RNAscope after staining requires the use of  mounting media to adhere a coverslip to tissue section or cell smear. This helps protect the sample and the staining from physical damage and helps improve the clarity and contrast of an image during microscopy.