Question

What is the difference between using GFP and immunohistochemistry, versus GFP and fluorescence?

Answer 1

The green fluorescent protein (GFP) is a protein composed of 238 amino acid residues (26.9 kDa) that exhibits bright green fluorescence when exposed to light in the blue to ultraviolet range.Although many other marine organisms have similar green fluorescent proteins,Green Fluorescent Protein (GFP) is a naturally fluorescent gene product of the jellyfish Aequorea Victoria.

As new drugs in development are geared towards gene therapy, GFP is being used as a reporter gene to ensure that the future therapeutic gene will be expressed in targeted cells. Therefore, monitoring GFP expression accurately is an important issue. The fastest and most cost-effective way to monitor GFP expression is by direct fluorescence microscopy to detect its autofluorescence. Other methods to detect GFP expression on histology slides are immunohistochemistry or immunofluorescence but they are more time-consuming and expensive. Autofluorescence intensity can be quenched by different parameters including the type of fixative used prior to the freezing procedure.

A period of fixation is necessary prior to freezing in order to retain the GFP, which is soluble, within cells; otherwise, the protein leaks out of cells that have lost membrane integrity. The fixation method of choice for GFP detection by direct fluorescence microscopy (i.e. to ensure good tissue quality and good fluorescence intensity) is by perfusion with 4% paraformaldehyde which is not always available in terms of equipment and cost. This poster compares two fixation methods by immersion and their results in terms of tissue quality vs GFP fluorescence intensity in rats.

GFP fluorescence was detected in all tissues with either fixative. Fixation time did not influence fluorescence intensity as the signal was similar for a given fixative. Comparing both fixatives, GFP fluorescence was more intense in tissues fixed in paraformaldehyde.

The down side of this fixative was a mediocre tissue quality: cells were shrunken and there were gaps within the tissueThis appearance was variable depending on each tissue’s fragility pancreas and liver structures being better preserved than other tissues. Tissue quality was well preserved using formalin/picric acid fixation and GFP fluorescence was detected even though less intense than with paraformaldehyde fixation Blocks were stored at -20˚C for 6 months and new sections were cut and mounted on glass slides to evaluate GFP direct fluorescence. It appeared that block quality was altered after long term storage.

Fluorescent dyes and proteins

Today, fluorophores are commonly used to label biological materials in nearly every life-science discipline. Fluorophores used in the life-sciences commonly fall into one of three categories:

• Fluorescent Dyes: Small, organic fluorescent molecules, either natural or synthetic, that can be used to label biologically relevant molecules. (e.g. Fluorescein, DAPI, DiI, Ethidium Bromide, Cyanine & Alexa Fluor™ Dyes)
• Fluorescent Proteins: Larger, biologically produced proteins, either natural or synthetic, that fluoresce as a byproduct of their macromolecular structure. (e.g. GFP, RFP, YFP)
• Quantum Dots: Nanoscale synthetic fluorescent crystals with an incredibly diverse range of uses.

The three classes of fluorescent probes possess similar properties, and many fluorescent proteins have been engineered to share a nearly identical excitation and emission profile to commercially available dyes (e.g. mutations to green fluorescent protein produced eGFP which is almost spectrally identical to the dye FITC). However, they are often applied in different scenarios.

Fluorescent proteins like GFP or RFP are commonly used to label the protein product of a specific transgene. Using molecular cloning techniques the coding sequence for a fluorescent protein is coupled to the coding sequence for the researcher's protein of interest. The resulting "fusion-protein" is a combined unit containing both the researchers target and the fluorescent protein. This powerful technique allows a researcher the opportunity to track the sub-cellular distribution and movement of their protein of interest in vivo.

Small fluorescent dyes, on the other hand, are most often used in in vitro experiments. Some dyes like DAPI, DiI, or Ethidium Bromide will associate with biologically relevant molecules or structures on their own, allowing them to be used independently to label these structures. Other dyes like Fluorescein, Cyanine, Rhodamine, or the wide variety of Alexa Fluor™ dyes are commonly used as conjugates for primary or secondary antibodies, which are used in immunolabeling experiments like:

• Immunofluorescence
• FACS/Flow Cytometry
• Western blotting

Cryomicrotome sectioning was difficult and resulted in mediocre tissue quality. Quality was still markedly better with formalin/picric acid fixation compared to paraformaldehyde. GFP fluorescence was detected in paraformaldehyde fixed tissues but the signal was much weaker compared to past sections . No fluorescence could be detected in formalin/picric acid fixed tissues after a 6-month storage. After approximately 1 year of storage, several blocks were sectioned and immunohistochemistry with an anti-GFP antibody could be performed with success

immunohistochemistry (IHC) is a powerful microscopy-based technique for visualizing cellular components, for instance proteins or other macromolecules in tissue samples. The strength of IHC is the intuitive visual output that reveals the existence and localization of the target-protein in the context of different cell types, biological states, and/or subcellular localization within complex tissues.

The IHC technique was invented during the 1940s (Coons, Creech, & Jones, 1941) and is routinely used as an important tool in health care and pathology for e.g. diagnostic purposes or to stratify patients for optimized treatment regimes. IHC is also widely used in research where molecules of interest are analyzed to study their roles in both healthy and diseased cells and tissues on the molecular, cellular or tissue level. There are many different ways to perform visualization of targets in tissues using IHC or IHC-based methods, and numerous protocols exist for different applications and assays. Even though IHC is generally a robust and established method, new assays often need careful optimization depending on the tissue or on the properties of the target protein, binder-molecule and/or reporter system. Many years of technical development and the hugely increased availability for specific binding-molecules have greatly improved the usefulness and areas of applications for IHC.

The progress in the field of IHC-based techniques and reagents has enabled scientists and health care providers with more precise tools, assays and biomarkers. In addition, technical advances have enabled e.g. highly sensitive simultaneous detection of multiple proteins in the same sample, and the detection of protein-protein interactions