*4.7. In Situ Hybridization*

In situ hybridization was performed using protocol and digoxigenin (DIG)-labeled probes established by Simmons et al. [37]. 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.
