Washing- and staining solutions, blocking agents, detection and mounting solutions. Successful immunohistochemical staining requires reliable, high-quality reagents to fixate, to wash and permeabilize the tissue. But what is the rationale behind the fact that certain buffers or storage conditions are better to use than other, or is it all just empiric?
Easier done than said
“What is the best blocking buffer for my immunofluorescence? Which antigen retrieval method should I use? PBS or TBS as washing buffer?” Well, these are all reasonable questions. Unfortunately, we don’t have a specific answer because it all depends on your target protein, the application you will use and the primary antibody you have chosen. The choice of buffers and diluents affects the stability and the binding properties of the antibodies and must be optimized for each specific antibody and experimental setup.
Years of research experience has taught us that immunohistochemical protocols, buffers and the choice of the antibodies “are seated in the walls” and belong to the lab history: they are selected far in advance and are not changed during the cause of long-term experiment or throughout a project that can last for several years.
This blog does not aspire to cover every aspect of the topic, but mainly to provide information that can assist in getting acquainted with the main essentials any user should be aware of directing the choice, manufacture and storage of buffers for immunohistochemistry.
The best solution for my IHC solutions?
During immunohistochemistry it is necessary to block all nonspecific binding sites within the tissue sample. This blocking step should be performed following the sample preparation, immediately before incubating the sample with the primary antibody. Commonly used blocking solutions are for example bovine serum albumin (BSA), and casein, which thought to prevent non-specific background by blocking hydrophobic interaction between proteins and ionic or electrostatic interactions.
Yet, casein, BSA, and dry milk can all be contaminated with bovine IgG which could be an issue for the use of certain antibodies. In fact, many secondary antibodies, like anti-bovine Ig Ab, anti-goat Ig Ab, and anti-sheep Ig Ab, will react strongly with bovine IgG. Therefore, the use of BSA, dry milk or casein as a blocking agent may actually increase background and reduce antibody titer.
You may also want to block adhesion of antibody to Fc-receptors on tissue macrophages and granulocytes. This is usually more of a problem in macrophage-enriched tissues like bronchioalveolar epithelia of the lung. To that end it can be advised to pre-block the tissue with Fab2-fragments of the antibodies (lacking Fc-domains). The simplest solution to get rid of this nuisance is to pre-incubate the section with a solution of 5% of non-immune serum from the same species as which the secondary antibodies were raised.
Is it advisable to add a surfactant, like Tween-20, in the washing buffer during antibody staining? Tween 20 is used in the washing steps to remove non-specific binding of the antibody. This is a crucial step in any fluorescence staining experiment since fluorochrome binding is highly sensitive, so tends to give background much higher than other methods. However, the choice of the detergent depends on your sample. You should know that Tween 20 contains lauric acid and is a very strong detergent. If you prefer a milder detergent opt for Tween-80 (polyoxyethylene sorbitan monooleate) which contains oleic acid instead of laurate. For cellular permeabilization of cell monolayers cultured on a coverslip, Saponin (0.1%) is the recommended choice since is not as harsh of a detergent. For brain slices, 0.2% Triton X 100 (stronger than Saponin) would be a typical choice.
Another example is the antibody dilution solution. The antibody diluent depends on the antibody itself. There is no a single recipe and the antibody data sheet provides you with that information. Antibody manufacturers frequently use phosphate buffer saline (PBS) or some similar phosphate-based buffer, usually at pH 7.2-7.4, often with sodium-azide added as preservative (to prevent microbial contamination). However, sodium azide is highly toxic a fact anyone should be aware about when using it on cell cultures.
TBS or PBS? This is the question.
Most biological processes are pH dependent. The purpose of a buffer in a biological system is to maintain a homeostatic intracellular and extracellular pH within a very narrow range and resist changes in pH in the presence of internal and external influences. The buffer used in each experiment depends on the requirements and limitations of the experiment itself. The best choice generally depends on your sample type, source of antibody, and detection method. Not all blocking agents and buffers are compatible with all antibodies and detection methods.
It is a common belief that phosphate buffer solution (PBS) should be the "all-purpose buffer”. However, choosing a buffer depends upon the objective of your experiment and the nature of your method (enzymatic reactions, histological procedure, live/dead cell detection methods, etc.).
PBS (pH 7.4) is usually used in cell culture, rather than TBS (Tris-HCl Buffered Saline). For immunoblotting/staining of phosphorylated proteins, TBS is recommended, since the phosphorus within the PBS interferes with the interaction between phosphorylated proteins and its cognate phospho specific antibodies. So, if your target is post translationally modified by phosphorylation (i.e.phosphorylated), you should go for TBS, not PBS.
In general, avoid PBS in any method that is sensitive to the presence of phosphates. Avoid TBS in any method that is sensitive to the presence of amines. Remember that any difference in the pH can affect the binding of your antibodies!
Phosphate buffer solution (PBS)
- Most physiological of common buffers mimicking certain components of extracellular fluids.
- Non-toxic to cells.
- pH changes insignificantly by temperature.
- Stable for several weeks at 4 C.
- Precipitation tends to occur in concentrated TBE buffer solutions (usually 10X) very soon after they are prepared. This can be prevented by filtering the solution using a cellulose acetate or cellulose nitrate filter (0.2 – 0.45 µm).
- Becomes slowly contaminated with micro-organisms
- Not to be use with alkaline phosphatase (AP)-based secondary antibody detection.
- PBS might create issues if you are looking at a phosphorylated epitope.
Tris buffer solution (TBS)
- High capacity at higher pH required for some tissues and some cytochemical procedures.
- Physiologically inert.
- pH changes with temperature. Must be measured at desired temperature.
- pH must be measured with certain type of electrode.
- Tris is a toxic polyamine, so not suitable to use for living cells experiments.
Buffers preparation: fresh or pre-made?
Most companies produce and provide pre-made, ready-to-use buffers for immunohistochemical, histological, cytological and biochemical applications enabling time saving procedures during the busy laboratory routine. Some laboratories prefer instead to prepare the buffers themselves. Whichever the option, the highest quality of all ingredients and the precision of the pre-set pH value must be guaranteed.
The commercial pre-made buffers are available in different formats:
- Ready-to-use buffers guarantee the largest level of reproducibility and ease of use without any additional requirements to the laboratory capacity.
- Concentrated buffers that must be diluted into a relevant working concentration and can be used directly afterwards. They are space-saving and have a longer shelf-life.
- Pre-mixed salts. These buffers can be stored at room temperature and are ready to use immediately after having dissolved in distilled water. They need the least storage capacity combined with easy handling and accuracy.
On the other hand, no matter the experimental conditions to be used, many IHC protocols suggest making fresh buffers. This choice has its own advantages. However, some buffers are around pH 7.5-8 when fresh, but with time their buffer capacity becomes compromised. Moreover, after a while some bacteria or fungi will grow.
To avoid these pitfalls, preparing concentrate stock of buffers is a good idea.
- A concentrated PBS solution is stable and can be kept for some time on the shelf or in the refrigerator.
- BSA (in PBS) is also a solution that you can keep for quite a while in the refrigerator without any problems.
- DAB, on the contrary, is very labile and must be made fresh each time.
Buffers storage: laboratory glassware or plasticware?
The market for buffers storage has been segmented as laboratory glassware and laboratory plastic ware. Currently, laboratory glassware accounts for the lion´s share of the global market. Although the glass-to-plastic lab ware conversion rate is increasing, you are strongly advised to ensure making informed lab ware choices matching your intended usage.
Some labs prefer the inert, heat-resistant, and customizable qualities that the glassware provides, while others prefer the price and the durability of plastic-ware. Plastic is flexible, cheap and easy to handle, whereas, glassware is fragile, expensive but best suited for conducting vigorous reactions and heating chemicals.
No glass is the same
By the way, did you know that not all laboratory glassware is the same? The composition of the glass can change significantly based on its country of origin due to its core component, sand, which can vary greatly from one country to another.
That's all for now. We hope you have found this article helpful! Don't forget to download our checklist with some useful tips on buffers and chemicals.
Buchwalow I, Samoilova V, Boecker W, Tiemann M. (2011) Nonspecific binding of antibodies in immunohistochemistry: fallacies and facts. Sci Rep. 1:28.
Stumptner C, Pabst D, Loibner M, Viertler C, Zatloukal K. (2019) The impact of crosslinking and non-crosslinking fixatives on antigen retrieval and immunohistochemistry. N Biotechnol. Sep 25;52:69-83.
Shi SR, Imam SA, Young L, Cote RJ, Taylor CR. (1995) Antigen retrieval immunohistochemistry under the influence of pH using monoclonal antibodies. J Histochem Cytochem. Feb;43(2):193-201.
Cătălin B, Stopper L, Bălşeanu TA, Scheller A. (2017) The in-situ morphology of microglia is highly sensitive to the mode of tissue fixation. J Chem Neuroanat. Dec; 86:59-66.
The "Laboratory Glassware and Plasticware Market: Global Industry Trends, Share, Size, Growth, Opportunity and Forecast 2019-2024" DUBLIN, May 8, 2019
Read the first part in the series: How to succeed with your IHC part I - Tissue Processing.