Frozen muscle samples were sectioned using a cryo-microtome (Leica-1850 UV) and Wisp1-mRNA expression in muscle cross sections was analyzed using the ViewRNA ISH Tissue Assay Kit and Fast red substrate following the manufacturer’s instructions (ThermoFisher Scientific, USA). Wisp1 was detected using the VIEWRNA type-1 probe-set for Mus musculus Wisp-1 (VB1-10640) and nuclei were detected with DAPI. Images were acquired using an Olympus-VS120 slide scanner. Image overlay with Pax7 was performed by merging the RNA FISH and Pax7 immunofluorescence on adjacent serial sections and overlapping morphological structures and selected DAPI-positive nuclei.
Fast red substrate
Manufactured by Thermo Fisher Scientific
Sourced in United States
Fast red substrate is a chromogenic substrate used for the detection and visualization of enzymatic activity in various applications, such as immunohistochemistry and in situ hybridization. It produces a bright red color upon enzymatic conversion, allowing for the localization of the target analyte.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using fast red substrate
1
Muscle Wisp1 Expression Analysis
2
Quantitative ISH for hUGT1A1 in Liver
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ISH was performed on formalin-fixed, paraffin-embedded liver sections using the ViewRNA ISH Tissue Assay Kit (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer’s instructions. Z-shaped probe pairs specific for hUGT1A1co were synthesized by the kit manufacturer. The deposition of Fast Red Substrate (Thermo Fisher Scientific) precipitates indicating positive signals was imaged by fluorescence microscopy using a rhodamine filter set. Sections were counterstained with DAPI to visualize nuclei.
To quantify RNA expression, five random pictures were taken from each liver section. Using ImageJ software (https://imagej.nih.gov/ij/ ), a threshold was determined to select UGT1A1-ISH-positive areas, and the percentage of positive area per image area was established. Likewise, an empty liver area (i.e., veins) was quantified in a second measurement. The final percentage of ISH-positive liver tissue (i.e., the percentage of positive hepatocytes) was calculated as the adjusted area (i.e., total area minus empty area), and the values were averaged for each liver.
To quantify RNA expression, five random pictures were taken from each liver section. Using ImageJ software (
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