Performing Double Fluorescence In Situ Hybridization to Detect Gene Expression in Mouse Brain Sections

Take a chemically-fixed mouse brain section.

Add a hybridization mixture containing a permeabilization agent and RNA probes labeled with FITC or DIG.

Incubate to allow cell permeabilization and probe entry.

The probes bind to complementary sequences on MBP mRNA, a reference marker ubiquitously expressed in oligodendrocytes, or ASPA mRNA, the target.

Wash to remove unbound probes.

Add a blocking buffer to prevent non-specific antibody binding.

Introduce HRP-conjugated anti-FITC antibodies targeting the FITC-labeled probe.

Wash, then add FITC-tyramide.

HRP oxidizes FITC-tyramide into a highly reactive form that binds to nearby proteins, amplifying the fluorescence signal.

Wash and reintroduce the blocking buffer.

Add alkaline phosphatase-conjugated anti-DIG antibodies targeting the DIG-labeled probe.

Wash and apply the Fast Red solution.

Alkaline phosphatase dephosphorylates Fast Red, producing a fluorescent precipitate.

Visualize under a fluorescence microscope.

Co-localization of Mbp and ASPA signals indicates ASPA expression in oligodendrocytes.

Dilute the two probes in hybridization buffer, pre-warmed to 65 degrees Celsius, and mix well. Apply 300 microliters of hybridization mixture onto each