Anti digoxigenin ap

Fluorescent and colorimetric RNA in situ hybridization (RNA-ISH) was performed on 7 µm paraffin sections as previously described (Paul et al., 2016 (link)). Complementary DIG or FL labeled RNA probes were generated following kit instructions (Sigma-Aldrich: 11277073910 and 11685619910) and were detected with Anti-Digoxigenin-POD (Sigma-Aldrich: 11207733910) and Anti-Fluorescein-POD (Sigma-Aldrich: 11426346910). For double fluorescent RNA-ISH the TSA Cyanine three and Fluorescein system from Perkin Elmer was used as directed (NEL753001KT). For colorimetric RNA-ISH Anti-Digoxigenin-AP (Sigma-Aldrich: 11093274910) was used to detect the probes.
Probes were generated to the following sequences:
Slides with fluorescence were mounted with Vectashield with DAPI (Vector Laboratories: H1200) and were imaged with a Nikon AZ100 Macroscope and photographed (Nikon Digital sight DS-Fi1). Fluorescent images were edited for contrast and color levels in Adobe Photoshop CS5.

In situ hybridization was performed using protocol and digoxigenin (DIG)-labeled probes established by Simmons et al. [37 (link)]. In short, cryo- or paraffin sections were thawed or deparaffinized and rehydrated, respectively. Sections were fixed using 4% paraformaldehyde (Merck, Darmstadt, Hessen, Germany; #818715) and treated with proteinase K (20 μg/mL, Merck, Darmstadt, Hessen, Germany; #1245680500). After another fixation step, sections were blocked. Probes (2 ng/μL) for PL1, PLF, and TPBPA were denatured and incubated with sections overnight, followed by a RNase A (AppliChem, Darmstadt, Hessen, Germany; #A2760) treatment. Sections were blocked, and anti-digoxigenin-AP (1:1000, Sigma-Aldrich, St. Louis, MO, USA; #11093274910) was added for probe detection. Incubation with BM-purple followed overnight; then, sections were counterstained with nuclear fast red (Sigma-Aldrich, St. Louis, MO, USA; #N3020). Sections were dehydrated, treated with xylene (VMP Chemie Kontor GmbH, Siegburg, North Rhine-Westfalia, Germany; #1000649172), and mounted in Entellan^® (Merck, Darmstadt, Hessen, Germany; #107960). Leica DM LB microscope (Wetzlar, Hessen, Germany) was used for analysis of sections.