Table 1.
PCR primers.
Figure 1.
Microcapillary electrophoresis of total RNA isolated from whole tibial epiphysis (A, C, E) or cryosections of tibiae (B, D, F) decalcified with 0.5 M EDTA (A, B), RNAlater/EDTA at pH 9.2 (C, D) or RNAlater/EDTA at pH5.2 (E, F).
Figure 2.
Total RNA extracted from tibiae decalicified with 0.5 M EDTA, RNAlater/EDTA at pH 9.2 or RNAlater/EDTA at pH5.2 was analyzed for Col2a1 (A) or Rpl10 (B) mRNA transcripts by q PCR using Sybr Green. The machine output (Roche LightCycler 480 II) of fluorescence at each PCR cycle number is shown.
Figure 3.
Cartilage morphology after EDTA or RNAlater/EDTA decalcification.
Tibial epiphyses were decalcified for 72 hrs at 4°C with 0.5 M EDTA (A) or RNAlater/10% EDTA at pH 5.2 and cryosections were stained with toluidine blue/fast green. The medial tibial plateau is shown. Cartilage morphology and aggrecan staining is preserved in the RNAlater/10% EDTA, pH 5.2 decalcified samples. Scale bar = 100 µm.
Figure 4.
In situ hybridization for Prg4 and Col2a1.
Cryosections from tibia decalcified with EDTA (A,B) or RNAlater/EDTA at pH5.2 (C–F) were hybridized with DIG-labeled Prg4 antisense (A,C) or sense (B,D) RNA probes, and against 35S-labeled Col2a1 antisense (E) or sense (F) RNA probes for Col2a1. Arrows show representative regions of target gene mRNA expression. Scale bars = 100 µm (A–D), 10 µm (E, F).
Figure 5.
Fluorescent immunohistochemistry of articular cartilage.
Collagen II (A, B) and collagen VI (C, D) protein localization was unaffected by RNAlater/EDTA, pH 5.2 decalcification (B, D) compared to conventional EDTA decalcification (A, C). Using both methods collagen II can be seen in both the pericellular and extracellular matrix, while collagen VI is predominantly localized to the pericellular matrix. DAPI was used as a nuclear stain. Scale bars = 50 µm.