In Situ Hybridization for Neurotransmitter Markers

Sense and antisense riboprobes were generated against full-length col19a1, syt 1, and syt2 IMAGE Clones (EMM1002-97504659 [col19a1]; MM1013-9199901 [syt1]; MMM1013-7512379 [syt2]; Open Biosystems, Huntsville, AL) or an 800-bp fragment of parv (corresponding to nucleotides 2–825) polymerase chain reaction (PCR)-cloned into pGEM Easy T vector (Promega, Madison, WI) with the following primers: 5′-TCT GCT CAT CCA AGT TGC AG-3′ and 5′-TCC TGA AGG ACT CAA CCC C-3′. Riboprobes were synthesized using digoxigenin (DIG) or fluorescein-labeled UTP (Roche, Mannheim, Germany) and the MAXI-Script In Vitro Transcription Kit (Ambion, Austin, TX). Probes were hydrolyzed to ≈ 500 nt. Coronal brain sections were prepared and hybridized at 65°C as previously described (Yamagata et al., 2002 (link)). Bound riboprobes were either detected by alkaline phosphatase (AP)-conjugated anti-DIG antibodies and colorimetric staining with the AP substrate NBT/BCIP (Roche), or detected by horseradish peroxidase (POD)-conjugated anti-DIG or anti-fluorescein antibodies and fluorescent staining with Tyramide Signal Amplification (TSA) systems (PerkinElmer, Shelton, CT). For double fluorescent in situ hybridization (D-FISH), after the first TSA reaction sections were washed with TBS, incubated with 0.3% H₂O₂ for 30 minutes, and reacted with the second POD-conjugated antibody. For fluorescent ISH coupled with immunohistochemistry (FISH-IHC) standard IHC was performed after completing the TSA amplification step described above. In some cases antibody-binding epitopes were destroyed during ISH, hampering IHC analysis. In such cases D-FISH was applied. However, most antibodies applied here were not hindered by ISH (see Supporting Fig. 1). Images were obtained on a Zeiss AxioImager A1 fluorescent microscope or a Leica SP2 scanning confocal microscope.