Alizarin red staining

For alizarin red staining, the fins were harvested 4 dpe, fixed overnight in 4% paraformaldehyde (PFA) in PBS and dehydrated in 100% methanol and stored at -20°C until use. Fins were rehydrated through a decreasing methanol series as described for ISH. Following that fins were bleached for 30 minutes in 0.8% KOH, 0.6% H₂O₂ (should be prepared and used within one week). Subsequently, fins were washed twice with water and then washed for 1hr in a saturated Alizarin Red solution containing 1% KOH [36] followed by 30 minutes wash in water and mounted in glycerol for imaging. The extent of mineralization was calculated as the ratio of the zone of mineralization (extent of detectable alizarin red staining length) to the total regenerate length. To evaluate the phenotypic effect of MO based knockdown experiments on alizarin red staining, the % similarity method was used as described above. The % similarity for each fin is calculated as ([ratio of {regenerate length: alizarin red staining length} of the injected side / ratio of {regenerate length: alizarin red staining length}of un-injected side] X 100). The mean of % similarity for the MO treated experimental group and the corresponding MM treated control group were estimated and compared, and the statistical significance between the groups was determined using two tailed unpaired student's t-test (P<0.05). For each experiment minimum of 6–8 fins were used per trial and 3 independent trials were performed.