Millane for successfully screening the protocol on (larval and adult) and em Hydractinia /em , respectively. which are based on strong digoxigenin (DIG) RNA and locked nucleic acid (LNA) probes, respectively. The combined IF-FISH process can be completed in 2 d for miRNA detection and 4 d for mRNA detection. TAK-901 TAK-901 Although optimized for is usually informative; however, regulation of RNA stability, localization and translation often results in differences in patterns of RNA and protein expression. In addition, localizing the expression of an RNA of interest to a given cell type is usually often challenging and can be facilitated by the use of cell typeCspecific markers to confidently identify and locate diverse cell types in complex tissues. Our laboratory is particularly interested in the regulation TAK-901 of stem cell behavior by intrinsic and extrinsic factors. The ability to conclusively identify germ-line stem cells and intestinal stem cells tissues. For example, embryos and imaginal discs have been quite amenable to ISH using standard protocols, whereas the cells within the testis have been quite difficult to analyze owing to the fact that this testis is surrounded by layers of muscle mass and pigment cells, which act as a barrier to probes. Therefore, the protocol explained here for dual labeling of RNA and protein was developed in the beginning to precisely determine RNA localization within cells comprising the testis niche7. Our protocol is based on several strong protocols previously explained to detect RNA in embryos8,9, imaginal discs10 and testes11,12, and miRNAs in zebrafish embryos13. However, as a result of the high annealing heat of the RNA probe, testis tissue became fragile and antigens recognized by well-characterized antibodies were not detected, complicating simultaneous analysis of RNA and protein. To overcome this difficulty, we optimized this protocol to perform IF under RNase-free conditions before exposing tissues to the harsh conditions necessary for FISH (Fig. 1). Open in a separate window Physique 1 Summary of steps involved in dual fluorescence detection of protein and mRNA/miRNA and the approximate time needed. Dashed boxes indicate that probe preparation is recommended at least 1 d before beginning the protocol. Gray boxes indicate the timing for miRNA detection. The modified protocol described below has been used to TAK-901 detect the expression of two important stem cell regulatory factors, ((in adult testes using LNA probes7. Even though dual protocol for mRNA detection is performed on dissected tissues kept in a microcentrifuge tube throughout the protocol, miRNA detection required mounting and fixation of testes on slides to enable better probe penetration (Fig. 1). Despite the fact that the protocol is designed for analysis of tissues, we predict that this approach (IF first, followed by FISH) should be relevant to tissues in other organisms, provided that the fixation conditions are optimized for the sample being processed. Open in a separate window Physique 2 Dual labeling of the stem cell niche in adult and larval testes. (a) Schematic showing the apical tip of the testis. Germline stem cells (GSCs) and cyst stem cells (CySCs) surround and are in contact with hub cells, which express (b) Detection of DIG-labeled riboprobe with the APCbased process. The AP substrate precipitates diffusely at sites of AP enzyme activity as shown by a homogenous stain (black) of hub cells11. (c) Detection of DIG-riboprobe (reddish) with tyramide transmission amplification (TSA) process. The peroxidase at the HRP-linked probe reacts rapidly with Tyramine, which results in a much increased spatial resolution of transcript detection. DAPI (blue) marks the nuclei of the cells at the apical tip of the testis. Note that the testis appears larger in c because of reduced tissue dehydration during processing compared with Foxd1 b. (dCf) Dual labeling with antibodies to the germ cellCspecific marker Vasa (d) and FISH for using a biotin-labeled.