Optimized RNA ISH, RNA FISH and protein-RNA double labeling (IF/FISH) in Drosophila ovaries

Journal name:
Nature Protocols
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Published online


In situ hybridization (ISH) is a powerful technique for detecting nucleic acids in cells and tissues. Here we describe three ISH procedures that are optimized for Drosophila ovaries: whole-mount, digoxigenin-labeled RNA ISH; RNA fluorescent ISH (FISH); and protein immunofluorescence (IF)–RNA FISH double labeling (IF/FISH). Each procedure balances conflicting requirements for permeabilization, fixation and preservation of antigenicity to detect RNA and protein expression with high resolution and sensitivity. The ISH protocol uses alkaline phosphatase–conjugated digoxigenin antibodies followed by a color reaction, whereas FISH detection involves tyramide signal amplification (TSA). To simultaneously preserve antigens for protein detection and enable RNA probe penetration for IF/FISH, we perform IF before FISH and use xylenes and detergents to permeabilize the tissue rather than proteinase K, which can damage the antigens. ISH and FISH take 3 d to perform, whereas IF/FISH takes 5 d. Probe generation takes 1 or 2 d to perform.

At a glance


  1. Workflow diagram for ISH, FISH and dual protein immunofluorescent staining and FISH (IF/FISH).
    Figure 1: Workflow diagram for ISH, FISH and dual protein immunofluorescent staining and FISH (IF/FISH).

    The arrows show the links between the steps in the three procedures.

  2. Comparison of permeabilization methods using ISH.
    Figure 2: Comparison of permeabilization methods using ISH.

    Shown are Stage 10B egg chambers. Anterior is to the left. Dashed lines indicate dorsal midlines. (ag) gurken mRNA localizes to the dorsal anterior region of the oocyte, between the nucleus and the oocyte membrane. (hn) broad mRNA localizes to two patches of somatic, dorsal, follicle cells that make up the dorsal-appendage primordia. Proteinase K permeabilization and DEPC treatment (a and h) compared with no DEPC treatment (b and i). (cg and jn) Comparison of alternative permeabilization methods, all using DEPC treatment, as indicated for detection of gurken mRNA expression: 15 min (cg, left images), 2 h (cg, right images); and broad mRNA expression: 45 min (jn, left images), and 5.5 h (jn, right images). Xyl., xylene; RIPA, detergents; Acet., acetone; No perm., no permeabilization. Scale bars, 50 μm.

  3. Optimization of dual protein and RNA analyses.
    Figure 3: Optimization of dual protein and RNA analyses.

    Shown are Stage 10B egg chambers. Anterior is to the left. Dashed lines mark the dorsal midline. (a,b) Performing the protein IF protocol before (a) or after (b) the FISH protocol affects the ability to detect broad mRNA (left images), β-galactosidase protein (middle images) and E-cadherin protein (right images). Permeabilizations were performed with xylenes and RIPA. Tyramide dilution was 1:50. Z-projections were rendered using ImageJ (a, left and middle, 5 μm; a, right, 1 μm; b, 6 μm). Identical adjustments were made in Photoshop for each pair of images being compared. (ci) broad mRNA expression in dorsal-appendage primordia. Identical adjustments were made to all images in Photoshop. (cf) Tyramide dilution was 1:50. (c,d) Permeabilization only with xylenes results in variable FISH detection: absence of signal in most egg chambers (c) and sporadic detection in a few samples (d). (e,f) Effect of timing of active-DEPC treatment on broad FISH signal strength and background. (e) Late DEPC: active-DEPC treatment on day 3 of the IF/FISH protocol. Laser transmission was increased to 20% to improve the signal strength. (f) Early DEPC: DEPC treatment on day 1 of the IF/FISH protocol. Laser transmission was 10%. (gi) Effect of tyramide incubation time on broad FISH signal. Permeabilizations were performed with xylenes and RIPA. Samples were incubated in a 1:100 dilution of tyramide for 15 min (g), 30 min (h) and 1 h (i). Scale bars, 50 μm.

  4. Detection of rare, moderate and abundant transcripts with ISH and FISH.
    Figure 4: Detection of rare, moderate and abundant transcripts with ISH and FISH.

    Anterior is to the left. Dashed lines indicate dorsal midlines. (ac) Low-abundance somatic transcript (mirror).(df) Medium-abundance somatic transcript (Paxillin). (gi) High-abundance somatic transcript (Rab40). (jl) Germline transcript (katanin 80). (a,d,g,j) ISH DIC images (projections were rendered using Helicon Focus software); (b,e,h,k) FISH laser confocal images, 25-μm projections of xy optical planes; (c,f,i,l) FISH, orthogonal views of egg chambers in b, e, h and k with 1-μm xy projections and 25-μm xz and yz projections. Note higher resolution and sensitivity of FISH compared with ISH as demonstrated by subcellular localization of nascent transcripts at genomic loci in germline cells (red arrowheads in l) and somatic cells (white arrowheads in c, f and l; inset in l shows a zoomed-in view of the follicle-cell nucleus boxed in white). Probes were generated for ISH and FISH in Peters et al.46. Information on tissue expression level is from modENCODE mRNA-seq data available on FlyBase. Scale bars, 50 μm.

  5. Optimized protein IF/FISH of Stage 10B egg chambers.
    Figure 5: Optimized protein IF/FISH of Stage 10B egg chambers.

    Anterior is to the left. Dashed white lines mark the dorsal midline. (a) Merge of bd: broad mRNA (green), α-Spectrin (magenta), bunched-lacZ reporter (blue). (b) broad mRNA expression in dorsal-appendage primordia detected by FISH. (c) Gradient of bunched-lacZ transcriptional reporter activity detected by immunofluorescent localization of β-galactosidase. (d) α-Spectrin immunofluorescence localization. (e) IF/FISH reveals distributions of broad mRNA (green) and E-cadherin protein (magenta). (f) IF/FISH detects the germline expression of gurken mRNA (magenta) and follicle cell expression of α-Spectrin. DAPI (blue) marks nuclei. Scale bars, 50 μm.

  6. Probe verification and quantification.
    Figure 6: Probe verification and quantification.

    All bands are at the expected size except where indicated. (a) Denaturing formaldehyde RNA gel. Lane 1: RNA Millenium marker. Lanes 2–8: probes used for ISH in Peters et al.46. Of the 20-μl transcription reactions, 1 μl is loaded in each lane. Lanes 2 and 3, nervana 3 antisense and sense (1.7 kb); lanes 4 and 5, shibire antisense (∼1 kb) and sense (difference in length due to position of restriction cuts); lane 6, shibire antisense, longer template (2.3 kb); lanes 7–8, huckebein antisense and sense (1.6 kb). Note the bands running faster than expected (arrows, aberrant bands; arrowheads, expected bands), possibly due to nuclease degradation of template DNA or RNA product or because of secondary structure in the template causing the polymerase to fall off prematurely. (b) Denaturing formaldehyde RNA gel. Lane 1: RNA Millenium marker. Lanes 2–3: 1 μl of 20 μl transcription reactions loaded in each lane for tiggrin antisense and sense probes (∼0.6 kb). Note the bands running slower than expected in lane 2 (arrows, aberrant bands; arrowhead, expected band), possibly due to incompletely linearized template DNA, which allows the RNA polymerase to make multiple processive cycles around the plasmid, yielding longer transcripts. (c) Dot blot. DIG-labeled antisense and sense probes against Drosophila shark comparing hydrolyzed and nonhydrolyzed (full-length) probes with DIG-labeled control RNA.


  1. Supplementary Video 1
    Video 1: Supplementary Video 1
    Drosophila Ovary Dissection. Supplementary video showing an example of the ovary dissection procedure, including preparation, a typical dissection, dissection variations and tool maintenance.


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Author information


  1. Department of Genome Sciences, University of Washington, Seattle, Washington, USA.

    • Sandra G Zimmerman,
    • Nathaniel C Peters,
    • Ariel E Altaras &
    • Celeste A Berg


C.A.B. supervised the project. C.A.B., N.C.P., A.E.A. and S.G.Z. designed the experiments. N.C.P., A.E.A. and S.G.Z. performed the experiments. C.A.B. and A.E.A. optimized the ISH methods; N.C.P., A.E.A. and S.G.Z. optimized the FISH methods; and S.G.Z. optimized the dual IF/FISH methods. N.C.P. performed dissections for Supplementary Video 1. S.G.Z. filmed and edited Supplementary Video 1. C.A.B. and S.G.Z. wrote the paper and N.C.P. and A.E.A. commented on drafts of the manuscript.

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Supplementary information


  1. Video 1: Supplementary Video 1 (52.01 MB, Download)
    Drosophila Ovary Dissection. Supplementary video showing an example of the ovary dissection procedure, including preparation, a typical dissection, dissection variations and tool maintenance.

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