Primer for Immunohistochemistry on Cryosectioned Rat Brain Tissue: Example Staining for Microglia and Neurons

The overall goal of this communication was to introduce immunohistochemistry procedures to the reader. For this, the example of double-labeling with Iba1 and Pan-neuronal antigens to observe microglia and neurons in paraformaldehyde perfused, sucrose cryoprotected, cryosectioned rat brain was used.

This technique can be adapted to serve endless purposes. An array of different antigens in a variety of tissue types such as, but not limited to brain, lung, liver, kidney and intestine can be visualized immunohistochemically with minor adjustments to the protocol. Immunohistochemistry allows for the direct comparison of multiple markers and can encompass up to four different antibodies in a single sample. The number of antibodies used in one sample is limited by bleed-through, which describes overlapping of fluorescent wavelengths. There is a limited fluorescent light spectrum, which consequently limits the staining combinations within a single sample (see Figure 6).

Figure 6: Color spectrum chart. Each fluorophore emits a specific wavelength of light when excited by a specific laser or filter. In order to effectively produce multi-labeled tissue, there should be minimal overlap between specific fluorophores conjugated to the antibodies. Although there are at least 10 different fluorophores available on the market, it is not possible to obtain clear results if all 10 are used. For example, triple-labeling with wavelength emissions at 405, 488 and 594 (A) would give clear labeling as there is minimal overlap, if any, between these wavelengths. However, if wavelength emissions at 488, 514 and 555 were used, the staining would not be easily interpreted as each antibody may be excited by the laser or filter meant for the other (B). This would give false co-labeling results. Online guides for fluorophore selection are available by searching for “fluorescent spectrum viewer”.

When double-labeling, it is best to choose primary antibodies made in different species, as both antibodies can be applied simultaneously. Double-labeling with two antibodies from the same species is possible, although technically difficult. When both primary antibodies are made in the same species, staining must be done sequentially, by completing the staining for one antigen before the second. Fewer issues will arise if the primary antibodies from the same species have different Ig classes (i.e. mouse IgG and mouse IgM or different subclasses IgG1 and IgG2a). For more details consult additional references^10,11. This protocol uses rabbit anti-Iba1 and mouse anti-Pan-neuronal. It is also necessary to include appropriate negative controls. For double-labeling with Iba1 and Pan-neuronal, one slide contains no primary antibodies, one slide contains anti-Iba1 only and one slide contains anti-Pan-neuronal only (see Figure 4). These negative controls confirm an absence of cross-reactivity and false staining. All other slides contain both primary antibodies. It is also important to avoid using secondary antibodies that may cross-react. For example, using donkey anti-rabbit 594 and rabbit anti-mouse 488 would result in donkey anti-rabbit 594 binding to both rabbit anti-Iba1 primary antibody and the donkey anti-rabbit 594 anti-mouse 488 secondary antibody. This would create false co-labeling.

Antibodies for a particular protein (antigen) are not always the same. For example, some antibodies are raised against antigens which are present in frozen tissue, whereas others will recognize an antigen which has been altered during the paraffin embedding process. For this reason, particular attention should be paid to the antibody specification sheet prior to purchase. If the intended application for the antibody is not listed, trouble-shooting may be required to obtain staining, or staining may not work at all. In addition, previously published data may offer “tricks of the trade” to obtain staining. Of importance to note is that subtle differences may be seen in different batches of antibodies; slight modifications may need to be made with each re-order of an antibody.

Most antibodies are good for 12 months from the date of receipt. Be sure to indicate the date of arrival on the product specification sheet. If antibody staining is not observed, the antibody may be too old or stored incorrectly. It is therefore also important to check each antibody’s specification sheet for proper storage.

Additionally, for many antibodies, different clones are available. Different clones recognize different parts of the antigen of interest. A given clone may be better for tissue subjected to different processing techniques (wax-embedded/frozen), target tissue type, or target species. Pay particular attention to the species in which the antibody was raised. When the antibody is raised in the same species as the tissue for analysis, high background may be observed. To combat this, additional blocking with commercially available kits such as a mouse on mouse kit, may be required for staining. This technique can also be adapted to cell culture. Fixation of cells, clone and specificity need to be checked on the specification sheet for applications.

Fixation with paraformaldehyde cross-links proteins, which could hide antigen binding sites in some conditions. Antigen retrieval can break protein cross-links and expose binding sites. If antigen retrieval is required, insert the following protocol after step 2.1.3: using a plastic staining dish and insert (see Table 1), microwave slides in citrate buffer on a low power setting for 10 minutes, taking care to not let the solution boil rigorously (specific power levels not included due to variability in settings between microwaves). Alternately, bring citrate buffer to 80 °C in a glass staining dish in an oven, then add slides for 10 min.

Poor tissue quality can also cause problems with staining (see Figure 5). There are a number of precautions one can take to avoid compromising tissue quality. First, be patient and diligent with sucrose changes in order to thoroughly displace water in the tissue before freezing. When freezing, keep the temperature constant. If the tissue freezes too slowly, water molecules will expand as they freeze, creating holes in the tissue. Alternately, freezing at too low a temperature will cause the tissue to crack.

Protocols will vary slightly for each antibody of interest. Upon receipt of a new antibody, one must optimize the protocol accordingly. If background is high (Figure 5), try blocking for a longer period of time. Alternately, increase the serum concentration of the block solution or add serum from multiple different species e.g. goat and rabbit serum. Additionally, casein can be used to reduce non-specific binding. Skim milk (made from dry powder) contains casein and can be used in a range of concentrations (typically, 1-5% w/v). Milk can be used alone, or in conjunction with serum. If an antigen of interest is an intracellular marker, detergents such at TritonX-100 or Tween-20 can be added to the blocking solution to permeabilize cell membranes. Thicker tissue sections can be processed similarly by floating the tissue sections in wells instead of mounting them onto slides. Paraffin-embedded tissue can also be processed similarly, but requires a de-waxing step prior to blocking. Feldengut et al.¹² addresses both of these methods.

Due to the light-sensitive nature of fluorescent antibodies, it is critical that fluorescent antibodies are stored in dark containers and avoid light exposure following secondary antibody incubation. In contrast, when using DAB as a developing agent, exposure to light is not detrimental. Fluorescent staining is limited in its longevity. Fluorescent-stained slides should be stored in a light tight box at 4 °C. Images should be captured within a month of staining. If immediate imaging is not possible, consider DAB or other alternative staining methods or delay staining until imaging is possible. Staining with DAB is usually limited to staining one target antigen, whereas fluorescence allows for double- and triple-labeling. Double- and triple-labeling can be achieved using DAB however this technique is prone to complications and difficulty in isolating specific antigen staining. DAB staining may be visualized under bright field microscopy, whereas imaging fluorescent staining requires a fluorescent microscope.

Another factor to take into account with DAB staining is that cells span multiple layers of tissue. It is therefore difficult to obtain clear images of cells such as microglia which have spanning processes. To overcome this issue, some investigators cut their sections thinner at 8-12 µm.

Imaging of fluorescently labeled tissue is best achieved on a confocal microscope. Confocal microscopes eliminate the haze normally seen with a research fluorescent microscope by isolating and capturing a single plane of focus within the sample. With confocal microscopy, finer details are sharper and bleed-through between fluorescent dyes is reduced. Thicker samples can be imaged one plane at a time (z-stack) to demonstrate true co-localization. When access to a confocal microscope is not available, imaging on a research microscope is possible, however exposure time and light intensity need to be locked between images.