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Fluorescent imaging training part 4: Troubleshooting ICC and IHC
Numerous factors can affect the outcome of your immunocytochemistry (ICC) or immunohistochemistry (IHC) experiments. To help you quickly find the cause of your problem and get things working again, we've compiled the most frequently asked questions (FAQs) and a troubleshooting guide.Overview
4.1 Troubleshooting ICC and IHC
4.2 Video troubleshooting guide
4.3 IHC FAQs4.1 Troubleshooting ICC and IHC
For IHC, to ensure that you get the data you need in your first experiment, we recommend our optimized kits and reagents:
• Buffers for heat-mediated and enzymatic antigen retrieval
• Highly sensitive labeled Streptavidin-Biotin LSAB ABC kits and micro-polymer kits for IHC detection and amplification (for mouse antibodies used on mouse samples, we recommend our Mouse-on-Mouse kit)
• Biotin and micro-polymer conjugated secondary antibodies to construct your own ABC method
• Optimized normal serums for blocking
Alternatively, you can try the tips below:No staining
The primary antibody and the secondary antibody are not compatible
Use a secondary antibody that was raised against the species in which the primary was raised (e.g., primary is raised in rabbit, use anti-rabbit secondary). Be sure that the isotypes of the primary and secondary antibodies are compatible (e.g., IgY vs IgG).
Not enough primary antibody is bound to the protein of interest
Concentrate the antibody more, incubate longer (e.g., overnight) at 4°C.
The antibody may not be suitable for IHC procedures which reveal the protein in its native 3D form
Check the antibody datasheet to see if it has been validated in IHC, and what type of IHC (formalin/PFA fixation, fresh frozen, etc).
Test the antibody in a native (non-denatured) western blot to make sure it is not damaged.
The primary/secondary antibody/amplification kit may have lost its activity due to improper storage, improper dilution or extensive freeze/thawing
Run positive controls to ensure that the primary/secondary antibody is working correctly (see section 'Using positive controls' for more information about controls).
The protein is not present in the tissue of interest
Run a positive control recommended by the antibody supplier (see section 'Using positive controls' for more information).
The protein of interest is not abundantly present in the tissue
Use an amplification step to maximize the signal. For example, use a biotin-conjugated secondary antibody and a conjugated streptavidin.
The secondary antibody was not stored in the dark (if your detection system is immunofluorescence)
Always prevent fluorescent-conjugated secondary antibodies from being exposed to light.
Deparaffinization may be insufficient
Deparaffinize sections for longer and use fresh xylene.
Fixation procedures (using formalin and paraformaldehyde fixatives) may be modifying the epitope the antibody recognizes
Use a different antigen retrieval methods to unmask the epitope (heat mediated with pH 6 or pH 9 buffer, enzymatic, etc).
Fix the sections for less time.
The target protein is located in the nucleus and the antibody cannot penetrate the nucleus
Add a strong permeabilizing agent like Triton X to the blocking buffer and antibody dilution buffer. See our protocol about permeabilization techniques.
The PBS buffer is contaminated with bacteria that damage the phosphate groups on the protein of interest
Add 0.01% azide in the PBS antibody storage buffer or use fresh sterile PBS.High background
Blocking of non-specific binding might be absent or insufficient
Increase the blocking incubation period and consider changing the blocking agent. Abcam recommends 10% normal serum of the species of the secondary antibody for 1 hr, or 1-5% BSA for 30 min for cells in culture.
Another option is to try a secondary antibody that has been pre-adsorbed against the Ig from the species of your samples.
The primary antibody concentration may be too high
Titrate the antibody to the optimal concentration, dilute the antibody further and incubate at 4°C (a slow but targeted binding is best).
Incubation temperature may be too highIncubate sections or cells at 4°C.
The secondary antibody may be binding non-specifically
Run a secondary control without primary antibody.
If you see staining with your secondary only, change your secondary or use a secondary antibody that has been pre-adsorbed against the lg of the species of your samples.
Tissue not washed enough, fixative still presentWash extensively in PBS between all steps.Endogenous peroxidases are active
Use enzyme inhibitors, ie Levamisol (2mM) for alkaline phosphatase or H2O2 (0.3% v/v) for peroxidase.
Fixation procedures (using formalin or paraformaldehyde fixatives) are causing autofluorescence (if your detection system is immunofluorescence)
Formalin/PFA usually exhibit autofluorescence in the green spectrum, so try a fluorophore in the red range.
Use a fluorophore in the infrared range if you have an infrared detection system.
Too much amplification (amplification technique)
Reduce amplification incubation time and dilute the secondary antibody or amplification kit.
Too much substrate was applied (enzymatic detection)
Increase substrate dilution and reduce substrate incubation time.
The chromogen reacts with the PBS present in the cells/tissue (enzymatic detection)Use Tris buffer to wash sections before incubating with the substrate, then wash sections/cells in Tris buffer.
Permeabilization has damaged the membrane and removed the membrane protein (membrane protein detection)
Use a less stringent detergent (e.g., Tween 20 instead of Triton X). Or simply remove the permeabilizing agent from your buffers. See our article about permeabilization techniques.Non-specific staining
Primary/secondary antibody concentration may be too high
Try decreasing the antibody concentration and/or the incubation period. Compare signal intensity against cells or tissue that do not express the target.
Endogenous peroxidases are active
Use enzyme inhibitors, ie Levamisol (2 mM) for alkaline phosphatase or H2O2 (0.3% v/v) for peroxidase.
The primary antibody is raised in the same species as the tissue stained (e.g., mouse primary antibody tested on mouse tissue). When the secondary antibody is applied it binds to all of the tissue as it is raised against that species
Use a primary antibody raised in a different species than your tissue. Use a biotinylated primary antibody and a conjugated streptavidin for the detection system.
The sections/cells have dried out
Keep sections/cells at high humidity and do not let them dry out.
Using positive controls
Why positive controls are necessary
To validate the staining in your sample, use a positive control. A positive control is a cell line or tissue section known to express the protein you are detecting. A positive result from the positive control, even if the samples are negative, will indicate the procedure is optimized and working. It will verify that any negative results are valid.
How to know what tissue type or cell line is a suitable positive control
First, check the antibody datasheet. This will often provide a suggested positive control. Always ensure the tissue or cell line you use is from a tested species.
If the antibody datasheet does not list a positive control, we recommend the following:
1) Check to see if there are any Abreviews for the antibody. Any tissues, cells or lysates that have been used successfully by these customers can be considered a suitable positive control.
2) Try looking at the Swiss-Prot or Omnigene database links on the datasheet. These databases will often have a list of tissues that the protein is expressed in. These can also be considered suitable positive controls.
3) Check the GeneCards entry for the protein. This will usually provide you with relative levels of expression in various tissues.
4) Check the Human Protein Atlas for the protein. This has a database of protein expression in different tissue types, cancers, and cell lines.
5) If you still have difficulty finding a suitable control, we recommend doing a quick literature search on PubMed to see which tissues and cells express the protein of interest.
Using negative controls
Why negative controls are necessary
Use a section from a cell line or tissue sample that is known to not express the protein you are detecting. This is to check for non-specific binding and false-positive results. Recommended negative control tissues are knockdown (KD) or knockout (KO) tissue samples.
No primary controls
This is when the primary antibody is not added to the sample. This indicates if any non-specific binding or false positives may be due to non-specific binding of the secondary antibody.
Antibody dilution buffer containing no antibody is incubated with the control sample in the same way as usual.
Isotype controls
An isotype control is an antibody of the same isotype (IgG2a, IgY, etc), clonality, conjugate, and host species as the primary antibody that is raised against a molecule that is not expressed in the sample you are using. Usually, this is raised against a chemical or a non-mammalian protein.
Use the same concentration (µg/ml) for the isotype control antibody and the primary antibody. This will determine the level of background in your sample.
Much like the no-primary control step, you would use this on your sample instead of the specific primary antibody. You would then use your secondary antibody as usual.
Controls for using a transfected cell line
We recommend to include an endogenous control if you are detecting a recombinant protein. This should be an essential part of the experimental plans.
There are inherent difficulties with antibody detection of recombinant proteins that need to be considered:
• Folding of the recombinant protein may be different from the endogenous native form. This folding may be preventing access of the antibody to the epitope. This is often the case with tagged proteins.
• Always ensure tags are placed on the N or C terminal end of the recombinant protein. • Always ensure the recombinant protein includes the immunogen sequence for the antibody you are using.
• An endogenous positive control is important to validate the results, as well as to indicate how well the reagents (eg antibodies) and procedure are working.
4.2 Video troubleshooting guide
If you'd like to quickly find out how to solve problems of no staining or high background, our two quick videos below highlight the major causes and what can be done to solve these problems.
No signal
High background
4.3 IHC FAQsWhich antigen retrieval method is most suitable for this antibody?
Any information we have available on a suitable antigen retrieval method will be stated on the antibody datasheet. If no information is available on the datasheet, we recommend starting with heat-mediated antigen retrieval using a citrate buffer. This will often require a certain amount of optimization by the end-user. You may need to optimize the time of antigen retrieval, or try one of the other available methods. Visit our article for more information on this and other antigen retrieval methods.
Which cell permeabilization method is most suitable for this antibody?
Solvents such as acetone and methanol are suitable for permeabilization when detecting intracellular proteins; however, they are not compatible with all antibodies.
Detergents such as Triton or NP-40 will also partially dissolve the nuclear membrane and are therefore suitable when access to nuclear antigens is required. However, they are harsh detergents that can disrupt membrane proteins, especially if left on for too long and are therefore not suitable for detecting membrane proteins.
Tween 20, Saponin, Digitonin and Leucoperm are much milder membrane solubilizers. They will create pores large enough for antibodies to pass through without dissolving the plasma membrane. They are suitable for antigens in the cytoplasm or the cytoplasmic face of the plasma membrane, and are also suitable for soluble nuclear antigens.
Why should I block endogenous peroxidase activity? Do I use PBS or methanol to make the H2O2 solution?
Endogenous peroxidases will react with the substrate solution (hydrogen peroxide and chromogen, e.g., DAB), leading to false-positives. This non-specific background can be significantly reduced by pre-treatment of the sample with hydrogen peroxide before incubation with HRP-conjugated antibody. Morphology of blood smears and peroxidase-rich tissues can sometimes be damaged by the hydrogen peroxide. Diluting the hydrogen peroxide in methanol is the best choice for fragile samples where preservation of morphology is required.
However, some cell surface protein markers are very sensitive to methanol or hydrogen peroxide quenching, reducing the staining of antigenic site, particularly on frozen sections. Using hydrogen peroxide in PBS is suggested for cell surface or membrane proteins. Another protocol modification is to quench with peroxide following the primary antibody incubation step.
Which fixation method is most suitable for this antibody?
The fixation and permeabilization method used will depend on the epitope and the sensitivity of the antibody, and may require some optimization. Fixation can be performed using crosslinking reagents, such as paraformaldehyde. These are better at preserving cell structures, but may reduce the antigenicity of some cell components, as the crosslinking may obstruct antibody binding (antigen retrieval techniques may be required). Another option is to use organic solvents such as methanol, ethanol and acetone. These remove lipids while dehydrating the cells. They also precipitate proteins on the cellular architecture.
The cells to be tested are growing in suspension. How can I stain these?
A suspension of bacterial cells or a cell line suspension can be spun onto a slide using a Cytospin™ centrifuge. This will leave a small circle of cell sample adhered to the center of the slide, which can be dried, fixed and stained.
What is the excitation and emission wavelength of the fluorochrome?
A list of fluorochromes and their excitation and emission wavelengths can be found here.
Can I do double/triple staining? If so, what should I consider?
In order to be able to examine the co-distribution of two (or more) different antigens in the same sample, a double or triple IF or IHC procedure can be carried out. Primary antibodies against two or three different target proteins can be used either in parallel (in a mixture) or sequentially.
Save time with pre-optimized kits
We recommend our double staining and triple staining IHC detection kits for a stress-free experience.
Alternatively, here are some key tips:
1. The primary antibodies must be raised in different species to ensure each one can be detected by a separate secondary antibody.
2. Each of the secondary antibodies used should be conjugated to a different fluorochrome or chromogenic chemical/enzyme. Each fluorochrome used should be excited by and emit a detectable wavelength with minimum overlap with one another. Chromogenic chemicals/enzymes used should each produce a different color. This will ensure that you can distinguish between the different target proteins in your sample.
We recommend optimizing the conditions for each antibody separately before trying them together.Summary
And that concludes the fluorescent imaging training series. We hope you now have a good idea of how to tackle most of the issues your imaging experiments might throw your way. Be sure to keep an eye on the abcam training page as new training series for different applications become available.