![]() When viewing the section using fluorescent microscopy the autofluorescence of the bone led to a high background signal (Figure 6). Contrary to the MTT assay, a preincubation at 4☌ did not improve the penetration of the dyes. Quantitative analysis of cores incubated with 1 μg/ml of each dye indicated that both dyes penetrated to approximately the same depth (400-550 μm) with cell tracker green penetrating further, although the difference was not significant. Both stains survived the embedding process. Whole cores incubated with ethidium homodimer 1 and cell tracker green were embedded in MMA and 6 μm sections were cut. It was observed that mountant containing DAPI could lead to the osteocyte lacunae appearing stained and would therefore be classed as viable ( Figure 5). The choice of mountant was determined to be important when visualising MTT stained cells. The marrow cells however produced a more diffuse staining (Figure 4A). Viewing the sections using fluorescence microscopy (Zeiss Filter set #10), which led to autofluorescence of the bone, did not improve viability determination. The punctuate pattern made recognition of stained cells difficult to determine under lens magnifications lower than 20x. This would appear to be the mitochondria surrounding the nucleus and as the mitochondrion is the site of MTT conversion such a staining pattern is to be expected. High magnification shows a ring of punctate staining within the osteocytes (Figure 4B). MTT staining of 250 μm thick sections for 4 h at 37☌ resulted in a good staining of all cells and led to good marrow preservation (Figure 4A). After decalcification and cryosectioning, the converted insoluble formazan is maintained within the viable osteocytes (Figure 3) but there is no maintenance of marrow structure. the core for 3 h at 4☌ followed by 3 h at 37☌ the whole core then demonstrated viable staining ( Figure 2).
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