Tibias from E18 mouse embryos (ICR line, Masaryk University) were dissected and placed in PSA (0.4g KCl, 8g NaCl, 0.35g NaHCO3, 1g glucose in 1000ml H2O, pH7.2, sterile filltered). Soft tissues were gently removed without disturbing epiphyses. Tibias were placed on Millipore filters above a metal mesh and cultured at air-liquid interface in F12/DMEM supplemented with 10% FBS, ascorbic acid (50 ug/ml), and 10 mM beta-glycerol phosphate for 8 days. Media supplemented with FGF2 (100 ng/ml), RBM-007 (100 nM), or scrambled aptamer (100 nM) were changed daily. Tibia were photographed and length of the tibias was measured from pictures by day 0 and at the end of cultivation (day 8) in Axio Vision (Zeiss). For histology, tibias were fixed in 4% paraformaldehyde (PFA for 24h), decalcified in 10% EDTA (pH7.2, 24h, two changes of the solution), washed thoroughly in DEPC H2O, dehydrated in ethanol series (70% EtOH for 1h, 80% EtOH for 1h, 90% EtOH for 1h, 100% EtOH for 1h), cleared 3x in xylene for 1h, paraffin infiltration 2x1h + ON , embedded in paraffin. Paraffin embedded samples were sectioned into 5um slices and placed onto microscopic slides and stained with hematoxylin and eosin. Col10a1 expression was detected on alternative sections by Mm-Col10a1 probe using RNAScope Technology (ACD Bio). The hybridized probe was visualized using the TSA-Plus Cyanine 3 system (Perkin-Elmer). DAPI was used to stain nuclei.

 

RNAScope is patented technology (acdbio.com). RNAscope Fluorescent Kit was used to detect Col10a1 and protocol was followed with no modifications (see attachement).