Non-radioactive in situ Hybridization Protocol Applicable for Norway Spruce and a Range of Plant Species
April 17th, 2009
We describe a modified DIG in situ hybridization protocol, which is fast and applicable on a wide range of plant species including Norway spruce. With just a few adjustments, including altered RNase treatment and proteinase K concentration, the protocol may be used in studies of different tissues and species.
Non-radioactive in situ hybridization protocol applicable for Norway spruce and a range of plant species. Carre Etal. Hi, my name is AM GaN, and we are working at OBS University and the Swedish University of Agriculture Sciences in obs Sweden.
We have established an inset visitation protocol that works on several different plant species. Today we are going to show you some demanding steps. In this protocol, we are going to show you embedding sectioning and different steps in the actual inset visitation experiment.
Let's go to lab One, fixation and embedding Step nine to 11 in the protocol. Step nine, preheat Petri dishes and molds at 60 degrees centigrade. Step 10, heat a pair of tweezers and distribute tissues evenly in a dish.
Add more histo wax so that the tissues are covered. Step 11, let the wax harden at room temperature. Two sexually steps 12 to 19 in the protocol.
Turn on the hot plates and have melted histo wax available. Take out the samples from the cold room. Step 13, use a scalpel to carefully cut out a wax block of tissue.
Shape the piece to facilitate slicing. Step 14, heat the block cold and the heel fuse them together. Let the prep harden that four degrees centigrade.
Step 15, cut the wax block into six to eight micrometer thick sections. Using a microtone sections are linked in one long ribbon. Cut up a ribbon and move it to paper.
At this point, it is possible to inspect the individual sections under a stereo microscope To choose sections of interest. It'll be easy to perform an inspection with tissues or stained with eoin. Step 17 at RNA free Milli Q water onto the slides.
Cut the ribbon into smaller pieces and put them on the slides. Move the slides to the hot plate. Step 17, allow ribbons to flatten out on a 42 degrees centigrade plate for one to two minutes.
Drain off the water using Kim oil. Step 19, incubate the slides at 42 degrees centigrade overnight so that the tissues adhere to the slides four in situ hybridization 4.1 in-situ section. Pre-treatment steps 36 to 48 in the protocol.
Step 36 depa sections in histo layer. The slides are evenly spaced and placed in a rack to facilitate the movement between different containers, dehydrate sections. In an ethanol series, add acetic anhydrate while stirring vigorously.
Step 45, set up for acetic anhydride treatment 4.2 in situ hybridization. Steps 49 to 52 in a protocol. Step 51, aging of pro one plus slides.
The solution is viscous. Try to avoid bubbles. Incubate the slides in a humid chamber.
4.3 init. Post hybridization, steps 53 to 72 in the protocol. Step 62, incubate the slides with the burer block in a plastic container.
Step 63, add enough blocking solution just to cover the slides. The antibodies are added to the slides using aging. Aging only works with prob bon plus slides For slides of a similar design, Step 65, allow capillary forces to pull up the anti dig antibody solution.
This step is repeated. Step 70, as before sandwich and draw up the western blue solution. Notice that the Western blue solution is drawn up twice, just as we did for the anti dig antibody solution.
And step 65, step 71 incubate slides in darkness for one to five days. At room temperature, the monitoring of hybridization signals can be done in a stereo microscope. While the slides are still sandwiched, it should be possible to observe purple to brownish stains.
This is what the Results from actually in CW station experiment look like. You can see the signal here as purple or brownish staining.
Overview
This article presents a modified DIG in situ hybridization protocol that is efficient and adaptable for various plant species, including Norway spruce. The protocol incorporates specific adjustments, such as changes in RNase treatment and proteinase K concentration, to enhance its applicability across different tissues and species.
Key Study Components
Area of Science
- Plant biology
- Molecular biology
- In situ hybridization techniques
Background
- In situ hybridization is a valuable technique for studying gene expression in tissues.
- Traditional methods can be time-consuming and species-specific.
- Modifications to existing protocols can improve efficiency and versatility.
- This study focuses on a protocol suitable for a range of plant species.
Purpose of Study
- To develop a faster and more adaptable in situ hybridization protocol.
- To demonstrate the protocol's applicability to Norway spruce and other plant species.
- To provide detailed procedural steps for researchers.
Methods Used
- Embedding and sectioning of plant tissues.
- Pre-treatment and hybridization steps for in situ analysis.
- Use of specific reagents and conditions to optimize results.
- Monitoring of hybridization signals under a microscope.
Main Results
- The modified protocol successfully facilitates in situ hybridization across multiple plant species.
- Adjustments in RNase treatment and proteinase K concentration enhance tissue compatibility.
- Clear visualization of hybridization signals was achieved.
- The protocol is efficient and reproducible for various applications.
Conclusions
- The modified DIG in situ hybridization protocol is a valuable tool for plant research.
- It allows for rapid and effective analysis of gene expression in diverse plant tissues.
- Future studies can build upon this protocol for broader applications in plant biology.
Frequently Asked Questions
Executive Industry Relevance
This protocol enables high-resolution spatial gene expression analysis in plant systems, supporting target validation in early discovery by confirming tissue-specific expression patterns. It reduces reliance on radioisotopes and extensive species-specific optimization, improving reproducibility and scalability for cross-species comparative studies. The method provides mechanistic de-risking value for phenotypic screening and translational biomarker discovery in plant-based biopharma pipelines.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Scientific Value: Confirms tissue-specific mRNA localization to interrogate therapeutic hypotheses and clarify gene function in reproductive development pathways.
- Operational Value: Enables functional target validation through direct visualization of gene expression in male reproductive organs across gymnosperm and angiosperm models.
- Predictive Value: Supports predictive confidence by demonstrating conserved expression patterns of homologous genes (e.g., AP3 homologs) in floral structures of Arabidopsis, Brassica, and Norway spruce.
Screening & Assay Development
- Assay Readiness: Prepares standardized tissue sections for downstream hybridization assays with consistent signal detection using DIG-labeled probes.
- Reproducibility: Eliminates RNase treatment and adjusts proteinase K concentration to enhance signal strength and reduce variability across species.
- Scalability: Facilitates high-throughput application across diverse plant species with minimal protocol adjustments, enabling platform reuse in comparative genomics.
Translational & Preclinical Research
- Translational Continuity: Demonstrates applicability from model angiosperms to gymnosperms, supporting extrapolation of findings to woody perennial systems relevant for biomass and biopolymer production.
- Mechanistic De-risking: Reduces ambiguity in gene function assignment by linking sequence homology to spatial expression in conserved developmental structures.
- Biomarker Alignment: Enables spatial correlation of gene expression with phenotypic traits in reproductive tissues, supporting biomarker discovery in plant molecular farming.
Pipeline & Workflow Integration
The method fits within the discovery biology phase, enabling hypothesis-driven target identification before lead compound screening in plant-based expression systems.
- Discovery Biology: Supports hypothesis testing by validating spatial expression of candidate genes in specific tissues and developmental stages.
- Screening: Generates quantitative spatial readouts that inform assay design for compound effect monitoring in transgenic or treated tissues.
- Analytics: Produces detectable chromogenic signals (purple to brownish) amenable to imaging and comparative analysis across experimental conditions.
- Translational Research: Connects gene expression patterns to developmental phenotypes in reproductive organs, supporting advancement decisions in trait validation pipelines.
- Enterprise Reuse: Establishes a standardized, adaptable workflow for cross-species gene expression analysis, reducing redundant optimization efforts.
Operational & Enterprise Impact
- Scientific Value: Increases target validation confidence through direct, spatially resolved mRNA detection without radioactive hazards.
- Operational Value: Enhances reproducibility by removing RNase treatment and standardizing proteinase K use, reducing protocol failure rates.
- Strategic Value: Accelerates go/no-go decisions in target selection by providing unambiguous expression data early in discovery.
- Portfolio Impact: Enables risk-adjusted prioritization of targets based on conserved expression patterns across species, improving success rates in downstream applications.
Implementation Considerations
- Requires expertise in histology, tissue embedding, and sectioning for plant materials.
- Dependent on access to microtomes, hybridization ovens, and chromogenic detection systems.
- Necessitates standardization of proteinase K concentration based on tissue density and phenolic content.
- Involves adaptation of incubation times and temperatures for different species and probe lengths.
- Limited by tissue autofluorescence and compound penetration in highly lignified or secondary metabolite-rich samples.
