Immunohistochemical Analysis in the Rat Central Nervous System and Peripheral Lymph Node Tissue Sections

Standard IHC procedures often require specific adjustments to obtain an optimal result, which commonly implies extensive experience but also "trial and error" approach. From tissue preparation until target visualization, almost each step in the protocol may be subjected to individually designed modifications in order to improve the final outcome. Double staining protocol presented here exemplifies IHC-based protein targeting particularly adjusted for assessing interrelations between the target proteins of our interest in formalin-fixed, paraffin-embedded rat CNS and LN tissue sections affected by neuroinflammation. As such, it may be used as a tutorial for learning the technique of IHC, but also as a source of inspiration for more advanced trials. However, it should be kept in mind that a mere reproduction of this protocol for targeting other proteins and/or in other tissues than here presented may generate suboptimal results.

In situ hybridization using locked nucleic acid (LNA) riboprobe against Ccl11 exhibited upregulation of the respective mRNA levels in CNS of the congenic rat strain¹⁰. This finding was confirmed by qPCR analyses. Further, qPCR revealed Ccl11 upregulation also in the congenic peripheral LN compared with the wild type (wt) strain. Finally, Western Blot (WB) showed significant upregulation of the Ccl11 protein in both target tissues of the disease-protected congenic rat strain¹⁰. Therefore, we aimed to investigate the origin of this chemokine directly in the target tissues, and for that purpose we developed the here described double staining protocol.

Besides avoiding mechanical damaging of the tissue during post-mortem dissection and post-fixation decalcification, tissue sectioning might appear quite challenging. The sectioning usually requires skill and practice. Quality of slice is dependent on many aspects such as adjusting a right angle between the tissue block and the knife in order to reduce the pressure applied on sample during cutting, cutting speed, etc. Thickness of the paraffin-embedded tissue slices produced by sledge microtome may vary from 2 to 50 µm. Cut paraffin tissue slices are mounted from a warm water bath onto glass slides preferably commercially coated with an adhesive such as poly-L-lysine or aminopropyltriethoxysilane (APTS) to ensure a better hold during the demanding staining procedure. In contrast to cryo tissue sections which are cut at approximately -20 °C using cryomicrotome and dried at RT prior to the staining, cooled paraffin blocks are sliced at RT, and subsequently dried at 50- 60 °C prior to deparaffinization.

Highly preserved tissue architecture, cell morphology and target epitopes are essential for assessing localization and interrelation of the target proteins. In order to achieve optimal tissue preservation, samples have to be properly fixed, using either coagulating or cross-linking fixatives. Coagulating fixatives, such as ethanol, are more often used for cryopreservation. Protein denaturation using ethanol is achieved through tissue dehydration⁹; which may result in inadequate cellular preservation¹⁶. In contrast to coagulating fixatives, formaldehyde is a cross-linking fixative which is most frequently used in routine histology and IHC⁹ for both cryo- and paraffin tissue preservation. We have achieved an optimal fixation for the rat CNS and LN by immersing into formaldehyde solution for 24 hr at 4 °C prior to decalcification and subsequent paraffin embedding. Despite causing profound changes in the conformation of macromolecules (formaldehyde namely forms methylene bridges that crosslink proteins and thereby mask antigens, which may prevent specific binding of antibodies), this semi-reversible, covalent reagent provides high quality tissue preservation. Notably, temperature and duration of fixation with cross-linking reagents may impact several aspects of IHC such as sectioning, epitope detection and even cross-reactivity. Thus both under-fixation and over-fixation using aldehyde-based reagents such as formaldehyde may cause artefacts that are primarily due to autolysis or excessive cross-links¹⁷, respectively. Nevertheless, an unspecific background staining due to hydrophobic protein binding observed by overfixation can be reduced using low salted buffers containing nonionic detergents, or by raising the pH of the dilution buffer when using polyclonal Abs¹⁸. However, an unspecific background staining (noise) caused by ionic and electrostatic protein interactions could be reduced using diluent buffers with high ionic strength, which at the same time may aggravate the noise caused by hydrophobic protein binding¹⁸. When it comes to detection of the target epitopes, formaldehyde-induced alteration of the tertiary structure of proteins can be, although not entirely, reversed by antigen retrieval¹⁹: i) by heating in buffers with various pH values (heat-induced epitope retrieval (HIER), ii) by enzymatic degradation (protease-induced epitope retrieval (PIER;²⁰)), iii) by incubation in strong alkaline or acid solution, or sucrose^21,22 or iiii) by treating with concentrated formic acid²³. HIER appears convenient for majority of the target epitopes¹⁹. Besides heating duration, pH value of the solutions such as EDTA (pH 5.5, 8.5 or 9.0) or sodium citrate buffer (pH 6.0- 6.2) may be essential for the outcome of HIER. Notably, the most satisfying result in our study was achieved by steaming the samples for 1h in the EDTA solution with the pH value 8.5.

Although antibodies preferentially bind to their specific epitopes, attraction to nonspecific, similar to the cognate Ag binding sites, cannot be entirely excluded. Notably, probably the most efficient method for reducing the background staining is incubation in blocking proteins (such as FCS used in our study) prior to application of primary Abs ⁹. Monoclonal primary Abs are anyhow preferred over polyclonal Abs due to higher specificity and thereby reduced unspecific background signal. Nevertheless, cross-reactivity still might appear, as monoclonal Abs are directed against epitopes commonly consisting of a small number of amino acids, which may be a part of some other (unspecific) protein²⁴. Unlike monoclonal, polyclonal Abs have a higher chance of recognizing multiple isoforms of the target protein. We initially used two different primary Abs to detect Ccl11: i) a polyclonal raised in goat and ii) a monoclonal raised in mouse. In our hands, both products exhibited the same staining pattern for the target chemokine. In order to perform simultaneous co-staining, we used goat-anti rat Ccl11 Ab in combination with the antibodies against Iba-1, Ed1 and Cd8, respectively, which were all produced in mouse. Nevertheless, besides simultaneously, double immunostaining can also be performed sequentially²⁵, as required for co-labeling of the primary Abs produced in the same species.

The optimal working concentration of the Abs used in our study was estimated through test-dilution series as an optimal ratio between intensity of the specific signal and the background noise. However, it should be kept in mind that an optimal working concentration of the same antibody may sometimes vary between organs, species, background pathology, etc. Notably, permeabilization was not required in our staining procedure performed in the paraffin embedded, 3-5 µm thick tissue sections, however, detergents like Triton X-100 could still have been used to reduce surface tension and thus to facilitate the binding between antigen and antibody. On the contrary, permeabilization- mediated improvement of Ab penetration is commonly required for protein detection in cultured cells or cell suspensions (immunocytochemistry (ICC)), for immunofluorescence (IF) in the cryo sections and IHC/IF in fresh (thick, vibratome-cut) free- floating tissue slices.

Multiple controls are essentially important for generating specific/reliable immunotargeting. As a positive control, we used lungs from a rat model of asthma, where we could detect Ccl11⁺ eosinophils. The negative controls were incubated only in the blocking solution o/n at 4 °C in absence of the primary Abs.

We have already mentioned some causes and potential remedies for reduction of a background signal. An unspecific immunostaining product may also occur as reaction between DAB and pseudoperoxidase of the red blood cells and peroxidase of the myeloid cells²⁶. Activity of these enzymes, just as the noise caused by endogenous biotin or AP has already been reduced during formalin-fixation. However, the pretreatment with methanol/H₂O₂ is necessary for their further or complete inactivation ^{27, 28}. Background staining caused by AP activity in mammalian tissues can be further inhibited by levamisole²⁹, or by acetic acid ²⁹. Unspecific binding as the result of ionic attraction between avidin and oppositely charged cellular molecules ³⁰ can be avoided by substituting avidin from egg white with streptavidin from Streptomyces avidinii ³¹.

DAB is probably the most frequently used chromogen in IHC. Besides DAB (brown reaction product), other peroxidase chromogens in use are 3-amino-9-ethylcarbazole (AEC; red), 4-Chlor-1-Naphthol (CN, blue) and tetramethylbenzidine (TMB; blue). Commonly chosen AP chromogens are FB, Fast Red (FR), new fuchsin and 5-bromo-4-chloro-3-indolylphosphate/nitro blue tetrazolium chloride (BCIP/NBT). The choice of enzymes and chromogens is basically a matter of individual preference, however, it may be steered by the target features such as localization, expression pattern, but also by the presence of endogenous pigments (melanin, hemosiderin)³². Counterstain is the last, facultative step in the IHC procedure usually performed using hematoxylin, nuclear fast red or methyl green¹⁸. Generally more suitable for the single labeling, counterstain facilitates the interpretation of tissue morphology and it should be subtly developed to avoid any interference with the chromogen precipitate.

Upon completion of the staining procedure tissue sections should be carefully mounted with a coverslip using a mounting medium. Formation of air bubbles should be avoided. Beside physical protection, mounting medium improves visualization and quality of imaging. Either organic/hydrophobic or aqueous/hydrophilic, the choice of the mounting medium is depending on solubility of the end product in the organic solvent. While a toluene- based mounting medium would be suitable, e.g., for DAB, aqueous mounting medium can be applied to all enzymatic chromogens, including fluorescence labeling.