Why is there a brown ppt in miRNA ISH ?

06 June 2016 0 3K Report

I did miRNA ISH experiment with 60nM probe concentration (Tm was kept at 55 degree), first time I visualized faint blue ppt . Second time I optimized some conditions like decreased Proteinase K concentration. After reading from online available troubleshooting guides, I changed substrate once in between the 2 hr of substrate binding time ( after 1 hr of incubation I changed substrate) . This time entire section turned brown. Why didn't the hybridization take place?

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