Immunostaining and In Situ Hybridization Lab Report

by savaria (student)

Immunostaining and In Situ Hybridization

Introduction and Theory

The study of gene expression provides invaluable insight into an organism's structure and function; how it is that from a mere embryo genes can control which cells, tissues, organs and limbs will develop, providing the organism with the inherited traits that specifically adapt it to its forthcoming environment. Mutations of particular genes are sometimes associated with certain birth defects. Should an organism with a mutation in just one gene be born with a specific birth defect, it gives a clue as to the gene's function. The question of its intended function may be better understood by discovering whether it became inactive during development to cause the defect. Of the collective amount of genes inherited by an organism not all are actively expressed ("turned on" and functioning). If expressed, it will be transcribed into RNA, specifically mRNA, providing the coding blueprint for a particular protein. When inactive and not expressed, no RNA is transcribed and thus no protein produced, perhaps because the protein is no longer needed or not required in a particular area of the body. To prevent the unnecessary expenditure of energy a core feature of many organisms was similarly evolved to inactivate genes that serve no current purpose.

Therefore a technique that enables the visualization of the internal embryonic environment will confirm whether RNA is present; its presence in an area is a good indicator that the gene coding for the RNA is performing a task required by the organism. In Situ Hybridization is just one biological technique that enables researchers to study such prenatal development. Specific probes are utilised to detect RNA. Due to its single-stranded nature, a complimentary length of single-stranded nucleic acids (the probe) is synthesized to hybridize through specific base pairing with the RNA of interest (see Figure One*). An embryo is placed in a solution containing this specific probe. The probe then locates the places in the organism where the RNA is present and hybridizes to it, hence the term "in situ hybridization" ('In situ' being Latin for 'in its original place'). The probe must be chemically labelled, whether by a radioactive label, a label that fluoresces under Ultraviolet light or one that can be detected by an antibody. This label, coupled with the probe, becomes the 'reporter' molecule, its visualization of which, enables localisation of RNA (or in fact DNA) sequences in heterogeneous cell populations. This includes tissue samples as well as environmental samples. Riboprobes even allow the researcher to localise and assess the degree of gene expression, particularly useful in neuroscience.

Figure One: Diagram to illustrate nucleic hybridization during In Situ Hybridization techniques.

Other applications of In Situ Hybridization include its use in microbiology, studying the morphology and population structure of microorganisms; the method is extremely sensitive and can detect the amount of mRNA contained in a single cell. Its application in profiling embryonic tissue (developmental biology) and establishing abnormal gene expression is reflected in other areas such as the profiling of pathogens in pathology. Furthermore, the technique is used in karyotyping and phylogenetic analysis due to the revolutionary ability Fluorescence in Situ Hybridization (FISH) had upon viewing chromosomal aberrations. Unique FISH patterns can be displayed even on individual chromosomes, showing defects. This is integral to a great deal of prenatal analysis, as it allows diagnosis of potentially fatal abnormalities before premature mortality occurs i.e. miscarriage. A suitable treatment can then be administered, or in some cases, defective development terminated. Physical mapping of clones on chromosomes is made possible because of this. We can directly assign previously-mapped clones to chromosomal regions associated with heterochromatin or euchromatin.

In Situ Hybridization is distinct from immunohistochemistry (IHC), which usually localizes proteins in tissue sections, rather than nucleic acid sequences. 'Immunostaining' was later developed as a term to encompass a wider variety of antigen-antibody staining methods used in histology and cell biology. The principle of this staining is that, by taking advantage of antigen-antibody (Ag-Ab) specific interactions, the presence of a specific protein in a tissue can be established, by using its known antibody to identify it. A specific monoclonal or polyclonal antibody is bound to the target immunogen before the Ag-Ab link is amplified (more true for 'indirect' immunostaining, see below) and visualized by an indicator system. Labelling with colouring (fluorescent) agents or electron-opaque substance (ultrastructural tags) beforehand allows such visualization. When using colouring agents, for instance, the emission of fluorescence when exposed to light of a particular wavelength (immunofluorescence) detects the Ag-Ab reaction.

There exists two means for utilizing immunostaining practices: a direct method and an indirect method. Following the evolution of IHC it has become typical to employ the indirect approach, which is not only cheaper but has the distinct advantage of producing a stronger signal. In the direct method there is no such amplification and therefore the Ag-Ab reaction appears very weak. It is a one-step staining process involving the labelling of one antibody which reacts directly with the antigen contained in the tissue section; it is simple and relatively fast. This method has since been improved, techniques adapted to progressively increase the sensitivity without the detriment of increasing the non-specific staining (background matter) as the lower levels of antigen are visualized. What is now commonly used as the successor to this method is the indirect technique for detection, which involves a primary unlabelled antibody, detected by a secondary antibody. The secondary antibody can be visualized with the aid of an enzyme conjugated to it (such as peroxidase, alkaline phosphatase or glucose oxidase; this is the indirect immunoenzyme method). With further secondary antibodies attaching to the primary (see Figure Two*), as well as even incorporating a third tertiary layer, the Ag-Ab signal is amplified even more with greater sensitivity. Concerning the three-stage methods, their early use did not label the second antibody and added an anti-enzyme antibody to which the enzyme had formed a complex.

Figure Two: Direct and Indirect Immunostaining

Today, more complex arrangements of antibodies and molecules detect antigenicity. Biotin and (strept)avidin in these complexes provide the best sensitivity and is used in most laboratories either as a conjugate or complex.

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Figure Two: Direct and Indirect Immunostaining

As well as correct antibody choice for the target antigens, full preparation of the samples is crucial to maintain cell morphology and the antigenicity of target epitopes (antigen component recognised by the paratope of an antibody). Often the sample tissue is perfused or blood-rinsed to prevent interference of hematologic antigens with those target antigens needing to be detected. Tissues are typically fixed in crosslinking reagents, (i.e. paraformaldehyde) before being embedded in paraffin to keep its natural architecture for storage. Tissue fixation immobilizes antigens while retaining subcellular structure, however because it chemically reduces protein solubility it may also mask target antigens if not done correctly, thus proper fixation is vital to ensure optimized results. Antigen retrieval techniques may be necessary should this occur, particularly if there is long fixation incubation or a high percentage of crosslinking fixative used. The solvent Xylene is commonly used then, to remove paraffin from IHC slides. Cells are permeabilized after formaldehyde fixation by covering the cells of the tissue with a permeabilization buffer temporarily (e.g. Diaminobenzidine solution, “DAB”). It should only be required for intracellular epitopes when the antibody required access to the intracellular matrix to detect the protein. However it is also required for detection of transmembrance proteins if the epitope is in the cytoplasm. Acetone, ethanol and methanol all permeabilize, though the latter is not always suitable. Permeabilization of tissues is also an important stage regarding In Situ Hybridization, enabling the probe access to the target. Embedding is a prerequisite for sectioning, should the decision to view the sample in sections be positive; whole-mount IHC allows visualization of even a whole organism, i.e. an embryo.

Finally, blocking is a technique used when a staining approach depends on biotin, peroxidases or phosphatases for amplification (or enzymatic antigen detection), whose quenching or masking of their endogenous forms prevents false positive and high background detection. Physical blocking of all endogenous biotin or chemical inhibition of all enzymatic activity will combat this. In reducing background staining blocking solutions are particularly effective, since they bind to any open protein binding sites that do not have attached any antibodies or antigens. Though they preferentially bind to specific epitopes, antibodies will also partially bind to a weaker degree on non-specific proteins at reactive sites, if they are similar to those of their target antigen's own binding sites. This non-specific binding significantly raises background staining and masks detection of the target antigen. Common buffers then, to incubate the samples with that blocks the reactive sites of proteins that the primary or secondary antibody may otherwise bind to weakly, include BSA, gelatin, non-fat dry milk or normal serum.

Immunostaining applications are as varied as they are numerous. Similar to In Situ Hybridization, it is most typically operated as an important diagnostic, predictive and investigative tool in histopathology for the diagnosis of abnormal cells. That is, in certain types of cancers, i.e. breast cancer. Specific molecular markers are characteristic of cellular events at one moment in the cycle, such as cell death (apoptosis) or proliferation. Particularly useful then, is IHC in developmental biology, when stages of development can be effectively frozen in time for examination. The exploratory potential concerning the cellular distribution of proteins, their absence or presence in combination with their intracellular location, as well as changes in protein expression or degradation, gives studies using immunostaining techniques a steep advantage over others. The staining can be carried out on a variety of different cells and tissues preparations, such as (as mentioned) formalin-fixed, paraffin wax-embedded material, frozen sections and cytological preparations. This allows antigens and their expression to be visualized in material that is under long-term storage. The results are permanent and so slides can always be reviewed at later dates, particularly useful for relating levels of protein expression in cancers to clinicopathological data of patient outcomes; comparisons can be drawn with further clarification of archived visual evidence.

Similarly to ICH, In Situ Hybridization (ISH) sample cells are treated to fix the target transcripts as well as increase the probe's available access. The probe hybridizes to the target complementary sequence at raised temperatures before all excess is washed clear. The temperature and other such controls as salt and/or detergent concentration may be altered to optimize the removal of non-identical interactions (only truly complementary sequences that match will remain hybridized). Then the labelled probe can be localized and its quantity measured in the target tissue using the most suitable method (e.g. fluorescence microscopy). ISH can use multiple probes to rapidly detect multiple transcripts simultaneously. Both ISH and IHC are methods that complement one another. For many genes, the sites where protein exists and can be visualized by IHC are not the same as the actual sites of synthesis, as revealed by In Situ Hybridization; often the protein is taken up elsewhere to target cells once it has been secreted. Both methods shed combined understanding on the activity and function of a gene and its different aspects of expression. ISH is a successful means for tracking down sites of potential synthesis the moment cloning is complete, far faster than the production of an antibody can be accomplished. Used in combination a comparison of the patterns of expression of mRNA and protein can be formed of the same histology section, widening understanding of gene expression in relation to its subsequent protein purpose.

Materials and Methods

Please refer to Practical Book for full list of Materials and for precise methodological timings.

Every incubation and wash step should be done on a rocking platform. Do not rock at high speed since the embryos are relatively fragile.

Immunostaining Procedure

Bleach the embryo in 4ml of 5% Hydrogen peroxide at room temperature for 30 minutes. Rinse with ethanol and rehydrate the bleached embryos through an ethanol series, dilutions of 50% and 30% incubated on ice with timings as shown in Practical Book. Detecting the target antigen with antibodies is a multi-step process requiring optimization at each stage to maximize signal detection. First dilute the primary (monoclonal mouse anti-neurofilament) and secondary antibodies (peroxidase-conjugated goat anti-mouse, Immunoglobulin G "IgG") into a buffer to stabilize them and promote uniform dissemination throughout tissue, also to discourage nonspecific binding (see Introduction for scientific theory). Such rinsing of the sample with buffer in between antibody applications is critical to remove unbound antibodies and also to remove antibodies bound partially (weakly) to nonspecific sites.

To do this, block the embryos using 10ml of TBST (Tris buffered saline with 1% milk powder and 1% Triton X100). Incubate on ice for 30 minutes and repeat this blocking wash and incubation twice more. Remove TBST and add 1.0 ml of diluted antibody to the experimental sample only. Leave overnight to incubate at 4°C. Afterwards wash the embryos with TBST three times, incubating after each wash at 4° C (initial two incubations at 1 hour each and the third overnight). The next day, again remove TBST and add 1 ml of the peroxidase conjugated second antibody. Incubate this overnight at the same temperature as previous incubation. Remove the second antibody and replace again with TBST to incubate for 1 hour. Repeat wash twice more leaving the final wash on overnight at 4°C. Remove TBST and add TBS and incubate in same temperature for 10 minutes before removing the TBS. Cover tubes with aluminium foil and add 1 ml DAB solution to incubate for 30 minutes in the dark. Remove DAB solution, add DAB + Hydrogen peroxide and monitor colour change. Discard solution and stop reaction by washing twice in TBS + 0.001% Sodium azide. Remove this and replace with methanol series of dilution at 50% 70% and 100% incubating each for ten minutes. Remove methanol and transfer to cavity blocks. Clear with mixture of benzyl alcohol. Cover with glass lid and incubate for 10 minutes. Observe neurofilament tissue under microscope.

In Situ Hybridization Procedure

Incubate tubes at Room Temperature (RT) in 50% methanol for 10 minutes after removing previous 70% ethanol. Replace with 20% methanol and incubate at RT for 10 minutes. Replace with PBT (Phosphate buffered saline + 0.1% Triton X100) and incubate for 10 minutes. Remove this PBT and replace with proteinase K solution; incubate at RT for 20 minutes. Wash twice with PBT before re-fixing embryos with Paraformaldehyde glutaraldehyde mix to incubate at 4 degrees centrigrade for 20 minutes. Rinse twice in PBT before incubating in hybridization solution at 62°C with a Parafilm overnight. After incubation, replace hybridization solution with hyb + control sense probe to control tube and hyb + FGF8 antisense probe to experimental tube. Hybridize overnight at 62°C. All next washings to be carried out at 62°C: 2 x SSC + 0.1% CHAPS to tubes to be incubated for 1 hour; 0.2 x SSC + 0.1% CHAPS to tubes for a 30 minute incubation and add KTBT to tubes before removing immediately. Incubate tubes with sheep serum in KTBT for 3 hours at 4°C before replacing with Diluted DIG –AP antibody overnight at 4°C in a rotating rack.

Replace antibody with KTBT to incubate for 1 hour on the rotator at 4°C. Repeat wash twice each for 1 hour duration. Add fresh KTBT and incubate overnight at 4°C on rotator. Next day replace KTBT with Alkaline Phosphatase buffer and incubate at RT for 10 minutes. Replace with Alkaline Phosphatase substrate, incubating at RT in the dark for 1 hour. Was twice in KTBT before replacing with 4% PFA in PBS for 15 minutes. Rinse with PBS twice. Replace with 25% glycerol for 10 minutes and follow through glycerol series of 50% 75% and 100% each for 10 minutes. Transfer embryos to cavity slip and observe staining using a stereomicroscope with base illumination.

Results and Discussion

Particular controls of temperature were used during both procedures, especially during incubations. For incubations of lower temperatures (i.e. 4°C) all tubes were kept in a cold room, a laboratory device used to standardize temperature for an extended period of time. For incubations of elevated temperatures tubes were kept sealed with Parafilm to prevent heat loss and stored in stable temperature-monitored incubation chambers.

Immunostaining Procedure

Organism used: 11.5 day old mouse embryos

Primary antibody: monoclonal mouse anti-neurofilament

Secondary antibody: peroxidase-conjugated goat anti-mouse IgG

Peroxidase substrate: Diaminobenzidine (DAB) + H2O2

Figure Three: Images of whole-mount embryonic tissue photographed after indirect immunostaining, control and experimental tissue

Above, in Figure Three, displays the image of the embryos from the experiment. The control had no antibody added to it and so was not expected to show any significant staining of tissue. However, perhaps most evident (and unexpected) is how not all ganglia and filaments are clearly visible. In both direct and indirect techniques there may be non-specific immunolabeling that can make it difficult to differentiate the correct target protein from others which bound partially to the antibodies; however in this experiment there seems to be significant problems in the staining. During tissue fixation, any delay or inadequacy may cause false positive results due to the passive uptake of serum protein and diffusion of the antigen. False positives are common in the center of large tissue blocks or throughout tissues in which fixation was delayed. This can be evidenced in the darkest regions of the embryo. Organic solvents could have been used as a fixative in place of paraformaldehyde, which remove lipids while dehydrating the cells; they also precipitate proteins on the cellular architecture. This may lower the "background noise" seen.

Some cells will contain endogenous peroxidase, the amount of which determined the tissue type. Endogenous peroxidases will react with the substrate solution (hydrogen peroxide and chromogen, e.g. DAB), leading to false positives. However this non-specific background was expected to be severely reduced during this experiment by the introduction of sample pre-treatment involving hydrogen peroxide before incubation with the conjugated antibody. The fact that there is still non-specific background is unexpected but may due to a variety of reasons. The secondary antibody may actually be too concentrated, requiring a decrease. The embryos may have dried during the procedure if left upon to be contaminated by the air too long, allowing the trapping of reagents under the edges of the specimen. Coupled with any inadequacies in the washings (at one time a washing had to be shortened to accommodate another laboratory group) these could easily cause background interference.

Positive control is to test a procedure to determine whether it functions correctly with reliable results. Ideally the known positive target tissue is stained, so that if this shows negative staining it sheds clarification on the fact that particular protocol measures need to be changed, until a positive staining is achieved. This experiment could therefore provide a good basis for a positive control test, confirming the need for the procedure to be checked. What is encouraging is that though heavily stained, there is at least staining. This means the antibody concentration was not too low. However what is more likely as an error is the over-staining of the embryo. The primary antibody concentration may have been too high, or its incubation too long. The tissue morphology of the embryo also appears to have deteriorated in appearance in places, perhaps in consequence of incomplete tissue fixation or over-digestion with the permeabilization solution. In such cases a tissue fixation protocol must be performed. Conversely the concentration of the Triton X100 could be lessened, or the duration of incubation in the permeabilization solution decreased.

In Situ Hybridization Procedure

Organism used: 9.5 day old mouse embryos

Probe: digoxigenin-labelled FGF8 antisense RNA

Antibody: alkaline phosphatase-conjugated sheep anti-digoxigenin

Alkaline Phosphatase Substrate: NBT + BCIP

The results are fortunately as expected; various developmental features are visible and were able to be labelled (see hand-drawn illustration in Practical Book), including the forelimb and hind limb buds, the bronchial arches, somites and midbrain/hindbrain boundary. In Situ Hybridization enables visualization in the location of mRNA and DNA in its native location. When detecting mRNA an "anti-sense" RNA probe is used. This is complementary to the mRNA sequence of interest in the target cell. Alternatively the "sense" probe will provide background signal and indicate the level of non-specific interactions. The control was given the hyb + control "sense" probe and not the hyb + FGF8 "antisense" probe and was therefore expected to receive no information regarding the mRNA location which is supported by the results. Both the "sense" and "anti-sense" signals are relatively detectable, and localized similarly in samples as the DNA is only denatured and has not changed its location in the cell.

Figure Four: Images of embryonic tissue photographed after In Situ Hybridization

Hybridization of the probe to the target sequence is affected by a number of influencing factors such as temperature and pH. From pH 5 – 9, the rate of renaturation is fairly independent of pH. Buffers containing 20 –50 mM phosphate, pH 6.5 –7.5 are therefore frequently used (PBS fulfilling that in this experiment). The agreed melting temperature is the point at which 50% of the two DNA/RNA strands are denatured and excessively unsuitable temperatures will cause any hybridization to be disrupted. To prevent this, temperature was controlled by specific incubation chambers with previously set temperatures determined to be at levels acceptable to maintain hybrid complexes. It can be seen from the image that other conditions were not optimal or checked, because the areas of targeted expression displays could be darker and contrast to a greater degree from the background. Observed in some studies is that 50% of hybrids are formed at the end of the first hour of incubation, perhaps eliminating the need for overnight incubation sessions. Washing should be undergone close to the stringency condition at which the hybridization takes place with a final low stringency wash, which it may not have been with the limited apparatus and usage of sieves when rushed instead of more accurate pipettes.

ISH experiments demand confidence in that the hybridization reaction is specific so that the probes are in fact binding selectively to the target mRNA sequence - not to other cell components however closely related to the target sequence. Ensuring that the correct probe is chosen and a high stringency conditions are maintained during washing, the probe should bind to the correct target sequence. Lack of mRNA expression in this experiment can be attributed to poor sample quality and/or RNA degradation rather than a true indication that no genes are being expressed. Embryonic tissue dries out if dehydrated too long and during washes, particularly when using sieves to transfer the embryo back into its tube; it was likely this occurred, as well as physical damage to the tissue itself. Some genes are continuously expressed such as Actin, and a probe specific to this will at least confirm whether it is the sample quality that is of fault. During hybridization duplexes form between both perfectly and imperfectly matched sequences, the extent seemingly affected by the stringency of the reaction. Unspecific background may be removed by washing the sample with a dilute solution of salt, the lower concentrations (and higher temperatures) of which increases stringency. Higher stringency is linked with greater specificity.

References

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GILBERT, S.F., 2006. Developmental Biology. 8th ed. Massachusetts: Sinauer Associates Inc.

JAVOIS, LORETTE, C., 1999. Immunocytochemical Methods and Protocols. Totowa, NJ: Humana Press Inc.

WIKIPEDIA, n.d In Situ Hybridization. In: Wikipedia: the free encyclopedia [online]. Available from <>. [Accessed 9th December]

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* Diagrams from Biology department power point presentations (BB20149 Molecular genetics of vertebrate development). University of Bath, Bath. Available at <>. [Accessed 28th November]

Biology Year 2 Molecular Genetics of Vertebrate Development