Basics of Immunohistochemistry

Immunohistochemistry is the process of detecting and visualizing antigens using antibodies. In principle, antibodies can be raised against almost any type of antigen. For the present guide, we will focus on proteins, which are among the most common antigens detected.

To visualize the antigen, one must:

1) Collect and fix tissue
2) Section tissue
3) Antigen Retrieval (if necessary)
4) Block Endogenous Enzymes
5) Incubate with a Primary Antibody
6) Incubate with a Secondary Antibody
7) Reveal the reaction with a reporter molecule


Lets explore these steps a little more:

1) Collecting and fixing tissue involves removing the desired tissue and using a chemical agent to preserve the tissue from decay.  In immunohistochemistry, paraformaldehyde  is often used to fix tissue because paraformaldehyde creates cross-links between proteins. Besides instilling mechanical stability in the tissue, it is also believed that the cross-links between proteins serve to trap other molecules that may be of interest (e.g. nucleic acids).

2) Sectioning tissue can be accomplished using a number of different methods. Common techniques involve embedding tissue in paraffin blocks, or embedding tissue in a freezing medium before slicing the tissue. When conducting frozen sectioning, it is often necessary to cryo-protect the tissue prior to sectioning. Sucrose is used as a common cryo-protectant as it is able to partially dehydrate tissue and prevent the formation of ice-crystals as the tissue freezes. Tissue is typically left in a 20-30% sucrose solution until the specific gravity of the tissue and the solution are approximately in balance, causing the tissue to sink. Sectioning then involves freezing the tissue by surrounding it with OCT or another freezing medium and using the freezing components of a microtome of cryostat to slowly freeze the tissue. A steel knife mounted on the sectioning apparatus is then used to repeatedly cut sections of a desired thickness.

3) For some staining techniques, antigen retrieval is necessary or can improve the quality of staining.  This process involves specialized reagents that can break the protein cross-links that have been formed by paraformaldehyde (or other fixatives) and uncovering biding sites that might have been hidden by these cross-links.

4) Endogenous peroxidases can exist in many cells or tissues. If present, peroxidases can result in a large amount of background staining that will not be specific to the antigen of interest.  One can test for the presence of endogenous peroxidase activity by incubating tissue with a DAB solution.  If the tissue turns brown, then the tissue contains endogenous peroxidase that that must be blocked.  Typically this type of blocking is done by exposing the tissue to a hydrogen peroxide solution. This reaction causes the irreversible inactivation of endogenous perodidase.

It is also necessary to ensure that the antibody for one's target antigen does not bind to non-specific binding sites.  To do this, these non-specific binding sites are saturated using a blocking agent.  Blocking agents like goat serum or BSA bind to portions of tissue with a net positive charge or portions that are hydrophobic. Without blocking, these areas would also serve to attract and bind most primary and secondary antibodies.

5) The next step in staining for a specific antigen is to incubate the tissue in the primary antibody.  This is as simple as diluting the antibody to a specific concentration and then allowing the tissue to agitate for a specified period in order to allow the primary antibody sufficient time to bind to the antigen sites.

One common question related to this form of blocking is how it is possible to block all of the non-specific sites on tissue and not block binding sites for our antigen of interest. The answer to this is actually quite simple: blocking buffers do in fact block antigen binding sites, but the types of bonds formed by the blocking buffer are very weak and can easily be reversed. Therefore, the high antibody-antigen affinity will always prevail over the bond between the blocking buffer and the antigen.

6) Similar to incubation of the primary antibody, tissue subsequently needs to be incubated with the secondary antibody. The secondary antibody is designed to bind to the primary antibody. Generally, this is accomplished by creating a secondary antibody that has been raised against immunoglobulin (IgG) for the species from which the primary antibody was derived.

7) The final step in an immunohistochemistry protocol is generally to reveal the antigen binding sites using a reporter molecule.  For fluorescent IHC, it is often the case that a fluorescent tag will be incorporated with the secondary antibody. Alternatively, signals can be further amplified by binding biotin molecules or an avidin-biotin peroxidase complex. The peroxidase in the complex is then revealed using DAB (3,3'-Diaminobenzidine). The oxidation of peroxidase using DAB and H202 results in formation of a free radical intermediate which polymerizes to form a brown stain. This reaction can also be turned black via the addition of Nickel to the reaction.

Hopefully this helps you to understand the steps involved in IHC. good luck staining!

D.J. Barker

Acoustics: Stimulus Presentation in the rat

There are a number of things to consider when presenting auditory stimuli to laboratory animals. One thing that we must not forget is that auditory stimuli are perceived differently by rodents than they are by humans. Thus, if we present a relatively low frequency tone to an animal, it is likely that their perception is different than the perception of the experimenter setting its intensity. As you can see from the collection of audiograms below (Engle & Barnes, 2012), stimuli below 1 kHz quickly fall outside of rats' hearing range but are still near the optimal range for humans.

Knowing this, imagine if you were to associate a 500 Hz tone with food delivery and a 2 kHz tone with sucrose delivery in a Pavlovian paradigm. Can you guarantee that the increased rate of approach for sucrose is in fact related to the increased incentive salience for that reward? Hopefully you would have learned about counterbalancing long before this, but if not, you can see how you might have a problem on your hands.

Another aspect of stimulus presentation that is often ignored involves the method by which we measure stimulus intensity.  Standard decibel (dB) meters that one might buy at your local electronics store or hardware store are known to measure the intensity of sound.  However, most individuals are unaware that the decibel measurements output by these meters are adjusted depending on their frequency.  We can see this in the plot below:

The weightings applied to commercial dB meters are designed to comply with OSHA standards so that acoustic hazards in work environments can be detected. Unfortunately, this means that the commonly available detectors are optimized/weighted for human hearing.  Thus, is is best to use a C or Z weighting on a decibel meter in order to obtain a more absolute measure of dBs and then to manually correct the volume to ensure that it is well above the auditory threshold for the rodent at that frequency.

All in all, being aware of these factors allows you to choose proper stimuli, assess stimuli effectively, or make adjustments to stimuli when necessary to ensure experimental reliability.  Similar concerns arise when considering any other sensory stimulus.  So, without a doubt, you need to be aware of the limitations of the species you are working with.

D.J. Barker

Perfusion/Fixation

A video on how to perform a perfusion by Gage et al. (2012)

This is a well conceived video on perfusion. It is important to note that the apparatus for perfusing buffers and paraformaldehyde differs from lab to lab. Still, the procedure described in this video is very sound and well described. Given the widespread use of perfusions in research, any researcher would do well to read about the principles behind the technique. Those interested should pursue other resources to learn more about the procedure.

Some relevant questions you might want to answer for yourself:

--What precautions must be taken when handling the chemicals used in this process?
--Why is paraformaldehyde used instead of formalin for immunohistochemistry?
--What is Sodium Azide? Why is it used? Are there any disadvantages to using sodium Azide?
--What is being preserved using this method? What is not being preserved?
--Name a few alternative approaches to retrieving neural tissue
--What is a good rule of thumb for the amount of fluid needed to pass through the body during a perfusion?


D.J. Barker

Renaming files in Matlab

A nice trick for renaming large numbers of files in Matlab:

Using Java code to rename files in Matlab

Correcting for Familywise Error

When conducting a group of statistical comparisons, it is important to understand the concept of familywise error or the 'familywise error rate'. The concept of familywise error describes the probability of incorrectly rejecting the null hypothesis. Perhaps more informally, familywise error represents the odds of reporting a statistical difference when no such difference exists.

 To understand why familywise error is important we must revert back to concepts learned in a basic introductory statistics class. First, we generally compare t-values (or other test statistics) to a known distribution of scores (t-scores in this case). Like any distribution, some values near the center of the distribution are more likely to occur than others. By understanding the odds that a given t-score will occur, we are then able to designate values that define regions of the distribution that are unlikely to occur as "critical values". That is, values above or below these critical values only occur with a certain limited probability.

 The exact probability we choose to define the critical values is arbitrary and often driven by tradition. As mentioned previously, we want to select values that are unlikely to occur in order to reliably report statistical differences. On the other hand, selecting values that are too stringent might cause us to report that no difference between our samples exists when in fact a difference does in fact exist. In psychology, a alpha of 0.05 is generally designated and denotes that falsely rejecting the null hypothesis should only occur 5% of the time. This value has been shown to provide a nice balance between selectivity and reliability.

Having said this, it is important to realize that the probability of finding a test statistic that is outside of the critical values for a single comparison is 1 in 20 (5%).  However, we also know from probability that the more chances one has, the more likely an event becomes. For example, if a player's chances of winning the lottery with one ticket are 1 in 14 million,  that player can increase his chances to 1 in 14,000. As you might imagine, this presents a problem when running multiple statistical comparisons.  The more statistical tests we run, the more likely it is that we will incorrectly identify a statistical difference (i.e. incorrectly reject the null hypothesis).

There are many ways to correct for this problem. Each different correction has its pros and cons and each correction may be more or less stringent than the next. Thus, one must consider how conservative of a correction a given situation calls for, if a desired correction will be overly liberal or conservative given the number of comparisons being made, and perhaps if the number of comparisons being made even warrants a correction at all.

One common correction that you might use is a Sidak correction.  This is a simple correction to calculate using the following formula:

 1−(1−α)^(1/n)

where, α represents your chosen alpha level and 'n' represents the number of comparisons being made.  This formula returns a corrected p-value that should be utilized in in lieu of alpha for each of your multiple comparisons when deciding whether or not to reject the null hypothesis. For example, if one were to make five separate statistical comparisons, the Sidak correction would dictate that only p-values of < 0.01 should be considered instead of the typical p< 0.05.

Try utilizing the formula yourself to calculate a corrected p-value and see how increasing the number of comparisons necessitates a greater correction.

D.J. Barker

Gas Chromatography explained

After our discussion today, I went looking for some tools to help illustrate the process for gas chromatography. Below you will find a schematic of the GC equipment and a great video by thermo scientific  demonstrating how a column works.

Techniques in Neuroscience

For those interested in a crash course in neuroscience techniques, I would highly recommend the book entitled Guide to Research Techniques in Neuroscience. I recently purchased this book and have been examining it most of the evening.  Techniques covered in the book include, but are not limited to:

-Imaging
-Animal Behavior
-Stereotaxic Surgery
-Electrophysiology
-Microscopy
-Genetics

This book will not necessarily give you the necessary skills to perform the techniques listed, but will give you a well-rounded overview of the techniques that might allow you to more effectively read, critique, and effectively discuss studies with techniques that are outside your purview. More importantly, this book provides a good middle ground between typical texts that are either oversimplified, overly-technical, or disconnected from the "hands on" side of neuroscience.

    D.J. Barker