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Protocol for Single-Molecule Fluorescence In Situ Hybridization for Intact Pancreatic Tissue.

STAR protocols (2020-10-29)
Lydia Farack, Shalev Itzkovitz
ABSTRACT

We describe an optimized smFISH protocol for the intact pancreas. The protocol is adapted from Lyubimova et al. (2013), a generic tissue smFISH protocol that works for most tissues but not the pancreas. The main changes implemented include increasing the period of mRNA denaturation from 5 min to at least 3 h and increasing formamide concentrations from 10% to 30%. These modifications yield sensitive single mRNA visualization that is comparable to those achieved in other tissues using the standard protocol. For complete details on the use and execution of this protocol, please refer to Farack et al., 2018, Farack et al., 2019.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Glucose Oxidase from Aspergillus niger, Type VII, lyophilized powder, ≥100,000 units/g solid (without added oxygen)
Sigma-Aldrich
D-(+)-Glucose, ≥99.5% (GC)
Sigma-Aldrich
Poly-L-lysine solution, 0.1 % (w/v) in H2O
Sigma-Aldrich
Dextran sulfate sodium salt from Leuconostoc spp., for molecular biology, average Mw >500,000 (dextran starting material), contains 0.5-2% phosphate buffer
Sigma-Aldrich
Catalase from Aspergillus niger, ammonium sulfate suspension, ≥4,000 units/mg protein