The RNA In Situ Hybridization core assists users with dissecting and freezing tissue (fresh or fixed) and cryostat sectioning (sections on slides or free-floating sections).
High-Throughput RNA In Situ Hybridization (ISH)
We perform RNA ISH (mRNA or miRNA) on a high-throughput platform (a Tecan EVO GenePaint liquid handling robot). This is a semi-automated, state of the art, liquid handling system modified with a flow-through technology for slide handling and sophisticated temperature control providing automation of approximately 90% of all manual steps in an ISH protocol. All steps of ISH – from pre-hybridization to additions of probes, hybridization, stringency washes and antibody-mediated chromogenic reaction – are carried out by the robotic system. This gives high-quality and reproducible results.
Although radioactive riboprobes were long considered to be the most sensitive way to detect mRNA, we are using a dual signal amplification system including digoxigenin labeled probes, anti-digoxigenin horseradish peroxidase labeled antibodies and tyramide amplification. This method achieves the sensitivity of radioactivity with added cellular resolution and it can be viewed with brightfield or fluorescence microscopy.
We currently use a Leica microscope equipped with a motorized stage and a CCD camera to create mosaic (stitched) images. This microscope will be replaced in December 2014 with a Zeiss Axioscan.Z1 for both brightfield and fluorescent imaging.
Figure 1. Equipment developed for automated in situ hybridization and illustration of typical results.
A. A flow-through hybridization chamber (150 µl volume) is composed of a standard size microscope slide with tissue sections, a pair of spacers and a glass plate with a depression. Slide, spacers and glass plates are fastened into a metal frame. Solutions are delivered by the liquid handling robot and initially flow through the chamber, but once the well is drained solutions are retained in a narrow slit, guaranteeing highly consistent staining results.
B. Hybridization and detection chemistry are performed on a Tecan EVO liquid handling platform equipped with a liquid handling arm with 8 pipetting tips for simultaneous liquid dispensing, racks for the flow-through hybridization chambers, temperature controlled reagent containers and high capacity liquid reservoirs for washing buffers, and an integrated thermal recirculator allowing high performance temperature control of the chamber racks. The instrument can process 192 slides simultaneously.
C. We currently use a Leica microscope equipped with a motorized stage and a CCD camera. This microscope will be replaced in December 2014 with a Zeiss Axioscan.Z1 for both brightfield and fluorescent imaging.
D. Sagittal brain section of an adult mouse showing the expression of the tyrosine hydroxylase gene which is expressed at numerous sites including olfactory bulb (OB), striatum (ST), cortex (CX), substantia nigra (SN), and cerebellum (CB). To visualize expression, digoxygenin (DIG)-labeled riboprobes are first detected with a anti-DIG antibody coupled with horse radish peroxidase. This enzyme is used to activate a tyramine-biotin conjugate, which thereby gets covalently attached to proteins in the vicinity of the anti-DIG antibody. Subsequently, biotin is detected with a steptavidin-alkaline phosphatase-based color reaction. The grid defines 32 images individually captured and then assembled into a composite image.
E. Blow-up of area boxed in D.
F. Expression of secreted frizzled related protein 2 (sFRP2) in the midbrain (MB) detected with a radioactive probe.
G. Expression of sFRP2 in the midbrain (MB) detected with a digoxygenin-tagged probe. Note the size of silver grain clusters on top of cells in F is broader than the size of a cell as defined by the color precipitate in G.
Microscopy and Automated Image Analysis
Traditional non-radioactive ISH produces a blue-colored precipitate that can be easily imaged in a bright-field microscope at magnification appropriate for detecting single cells. Using a microscope with a motorized stage, multiple images are collected from the same section (gray boxes in Fig. 1D). Individual images are stitched together to produce a mosaic representing the entire section. Starting in January 2015 the Core will start using a new scanner (Zeiss Axioscal.Z1) that is capable of both brightfield and fluorescent imaging.
We can also assist with quantification of non-fluorescent ISH (cell counts and expression levels) using software developed for the Core by Dr. James Carson (Carson JP et al. (2005) J. Microsc. 217:275-281).
Figure 2. Automated image analysis and quantitation of gene expression
Top panel: Left: sample expression pattern of Ly-6/neurotoxin homolog (Lynx1) in a sagittal section of adult mouse brain. Right: automated intensity analysis of the expression pattern, cells expressing high levels of Lynx1 are shown in red, medium expressing cells in blue and low expressing cells in yellow. Lower panel: Quantification of differences in Lynx1 expression strength between wildtype (wt) and barrelless (brl) mice in the hippocampus. Left: hippocampus CA2 region marked in yellow, CA3 region in purple. Middle: close up view of the Lynx1 expression pattern in the hippocampus in wt and brl mice and below the respective intensity analysis. Right: plot of percentage of cells expressing Lynx1 at high (red), medium (blue) and low levels (yellow). The automated intensity analysis was performed over three different sagittal sections from wt and brl mouse brains. The data clearly indicate a decrease of expression strength in the hippocampus of brl mice compared to wt.