Immunohistochemical analysis of protein expression in formalin fixed paraffin embedded human intervertebral disc tissues

Abstract Immunohistochemistry (IHC) is a useful technique for the localization and semiquantification of protein expression within tissues. Adult human intervertebral disc (IVD) tissues contain a large amount of auto‐fluorescence which often makes immunofluorescence techniques inappropriate on tissue samples but can be applied to isolated cell samples. Thus, IHC remains one of, if not the most common application for protein detection within IVD tissue. Immunostaining localizes antigen expression through specific epitope‐antibody interactions. Within the field of IVD research, IHC is commonly used on fresh frozen and paraffin embedded tissues to elucidate the expression of antigens. Here, we discuss the principles of IHC applied to formalin fixed paraffin embedded IVD tissue and supply optimized protocols for antibodies used within our group to guide research within the IVD field.

Following fixation in 10% w/v formalin or 4% (w/v) paraformaldehyde (all reagent suppliers and product codes listed in Table 2). It is essential to ensure appropriate fixation; uneven fixation will result in unreliable IHC staining and lead to difficulties in troubleshooting.
Over fixation may lead to brittle tissue sections. During fixation it is important to ensure sufficient volume of fixative to sample with a recommended 50:1 ratio of fixative to tissue sample. 2 Small fragments of disc tissue obtained from surgery such as discectomy, can be fixed within 48 hours while large whole IVDs should be fixed for a week to ensure complete fixation. For large samples where 50:1 ratio of fixative is not practical, the fixative should be changed every 48 hours, until fixative remains clear.

| Decalcification
If vertebral bone is included with the IVD sample such as cadaveric discs, a form of decalcification may be required. Decalcification of bone can be achieved using many different reagents that require varying lengths of time to fully decalcify specimens. Neutral 20% (w/v) EDTA (pH 7) requires a longer decalcification time but causes little tissue damage, whereas acidic decalcification acts rapidly but exceeding the end-point will lead to extensive tissue damage and a loss of IHC staining intensity and thus EDTA decalcification is recommended. 3 Complete decalcification should be confirmed by X-ray analysis prior to embedding into paraffin wax. Decalcification time should be optimized according to the size of the tissue sample.

| Embedding and sectioning
Following paraffin embedding thin sections (normally 4 μm thick) are prepared on a microtome and floated on a water bath prior to mounting onto positively charged slides (it is important to use thin sections to ensure antibody penetration). When mounting, extra care should be taken to avoid tissue folding. Where folds are generated these areas of tissue should not be included in downstream analysis as trapping of reagents may lead to nonspecific staining. Following mounting onto slides, sections should be dried for a minimum of 1 week to prevent detachment during staining (longer if heat antigen retrieval methods are to be used).

| DEWAXING AND REHYDRATION
Tissue sections embedded in paraffin wax should be deparaffinized in xylene or a xylene substitute such as Sub-X or Histoclear (Leica) prior to removal of xylene in alcohol (Industrial methylated spirts used here).
F I G U R E 1 Principle of Immunohistochemistry. Target antigens are detected by the application of specific primary antibodies, followed by the addition of a biotin conjugated secondary antibody which recognizes a specific sequence on the primary antibody. Localisation of bound biotinylated secondary antibody is detected by the addition of streptavidin HRP labeled ABC solution which binds to the biotin on the secondary. Enzymatic color changes occur when DAB reacts with the HRP bound to the secondary antibody, thus resulting in the detection of the target antigen

| ANTIGEN RETRIEVAL
As formalin fixation results in crosslinking of proteins this can lead to antigen masking. Furthermore, antigens can be hidden by extracellular matrix components or cellular membranes in the case of intracellular proteins. Therefore in many cases it may be necessary to unmask the antigen by performing an antigen-retrieval technique. 4 There are multiple antigen retrieval methods available which can be divided into chemical, enzymatic and heat retrieval methods. 5-7 Antigen retrieval methods are determined for each individual antigen and antibody; no antigen retrieval is also tested during optimization as this can also produce sufficient staining for some antibodies and antigens. Here, we describe the key methods utilized in the protocols for IVD tissue within our laboratory. It is our experience that these methods work reliably for IVD tissues, heat retrieval can be problematic leading to tissue section lifting and should only be used when necessary. It is recommended to leave sections to dry for longer periods if heat retrieval is required, steaming methods can be particular problematic for disc tissue as they often lead to tissue swelling and dissociation from slides.

| Enzyme antigen retrieval
Sections (in a slide rack) are placed in TBS containing 1% (w/v) α-chymotrypsin and 0.1% CaCl 2 (preheated to 37 C) and incubated for 30 minutes at 37 C. IHC Protocol for formalin fixed paraffin embedded IVD tissue all suppliers are provided in Table 2 Step

| ANTIGEN DETECTION
Action Timings  Graphical representation should show all data points or utilize a box and whisker plot or other graph suitable for nonparametric data.
For the quantification of extracellular proteins such as extracellular matrix markers cellular immunopositive staining is still useful as this indicates current cellular production of these proteins (as long as the antigen detects the intracellular form). This can be complemented by percentage area of immunopositivity which can be quantified using ImageJ software. The use of image intensity for IHC analysis should be avoided for ABC/DAB methodology as the signal is an amplified signal and intensity cannot be directly related to quantity of protein.

| Automated IHC analysis
While there are a number of automatic image analysis systems which enable automatic IHC quantification for tissues, which is particularly used in the cancer fields. 8 These systems can be problematic for disc tissues.
Disc tissues contain few cells (4 × 10 6 cells/cm 3 ) in the NP, 9,10 which means either large numbers of images must be captured and analyzed, or full slide analysis is required. For the majority of laboratories who do not have facilities for rapid reliable full slide image capture it is often more time and cost effective to manually quantify immunopositivity down the microscope. Furthermore, it is important to accurately identify tissue type of interest (eg, NP, AF, and CEP) during analysis and this can be just as time consuming to "label" tissue areas within image capturing software.

| STATISTICAL ANALYSIS
As data is not normally distributed (percentage data) data should be analyzed using nonparametric testing dependant on the groups and hypothesis being tested. For example, where investigating immunopositivity between grades of histological degeneration if more than two groups are analyzed a Kruskal-Wallis test with appropriate posthoc analysis can be performed when a significant difference is seen between study groups. Linear regression analysis can be used to observe correlations between for instance, percentage immunopositivity and grade of degeneration, and correlation between target molecules or age.