A Simplified In Situ Hybridization Protocol Using Non-radioactively Labeled Probes to Detect Abundant and Rare mRNAs on Tissue Sections

bstract The article describes a simplified and efficient protocol for non-radioactive in situ hybridization experiments on tissue sections. The sensitivity of the method allows the expression of genes to be studied with transcript levels ranging from very low (20–30) to high (several thousands) copy numbers per cell. The immediate procession of cryosections to the ISH protocol after sectioning, coupled to the active DEPC treatment of slides before hybridization, are shown to improve considerably the mRNA detection. Moreover, we also improved the detection of rare transcripts such as PPARα mRNA by increasing the hybridization time up to 40 h. The resolution obtained is such that distribution gradients of mRNAs present at less than 30 copies within the cells are detected, both on cryosections and paraffin sections. A Simplified In Situ Hybridization Protocol Using Non-radioactively Labeled Probes to Detect Abundant and Rare mRNAs on Tissue Sections