To obtain frozen sections, fixed brains were cryoprotected in 30% sucrose in PBS and embedded into Killik O.C.T. (Bio Optica, Milan, Italy) and then placed at −80 °C for quick freezing. Brains were sectioned into 12 µm-thick slices using the HM525 NX cryostat (Epredia, Portsmouth, NH, USA) and finally collected onto polarized SuperFrost™ Plus Adhesion Microscope Slides (Epredia).
ISH on frozen tissue sections was performed as described in Gabellini et al. [25 (link)], with some modifications. Cryosections were thawed and washed in PBT (PBS + 0.5% Triton X-100) and incubated with either 300 ng/mL prr21a-201 or prr12b-201 antisense probes at 65 °C O/N. Slides were then rinsed at 65 °C in Hybe Wash and MAB + 0.1% Tween20 (Sigma-Aldrich) (MABT) solutions at RT. After 1 h-long equilibration at RT in the previously described blocking solution added with 20% lamb serum, slides were incubated with anti-DIG antibody (diluted 1:2500 in the blocking solution) at 4 °C O/N. Slides were stained in BM Purple Solution in the dark at RT. After the staining procedure, images were acquired using a Nikon Eclipse Ti microscope (Nikon Instruments).
ISH on frozen tissue sections was performed as described in Gabellini et al. [25 (link)], with some modifications. Cryosections were thawed and washed in PBT (PBS + 0.5% Triton X-100) and incubated with either 300 ng/mL prr21a-201 or prr12b-201 antisense probes at 65 °C O/N. Slides were then rinsed at 65 °C in Hybe Wash and MAB + 0.1% Tween20 (Sigma-Aldrich) (MABT) solutions at RT. After 1 h-long equilibration at RT in the previously described blocking solution added with 20% lamb serum, slides were incubated with anti-DIG antibody (diluted 1:2500 in the blocking solution) at 4 °C O/N. Slides were stained in BM Purple Solution in the dark at RT. After the staining procedure, images were acquired using a Nikon Eclipse Ti microscope (Nikon Instruments).
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