**4. Lab procedures to determine Fb-3**

#### **4.1 Fb-evaluation from tissues**

Immunohistochemistry (IHC) is a laboratory technique, which enables to highlight the creation of antigen–antibody complexes inside a tissue.

Such diagnosis technique exploits the ability of some antibodies to recognize cellular proteins (like Fb-3), called antigens, which in tumoral cells may have expression characteristics (more or less apparent) other than those of ordinary cells. The sample, after fomalin paraffin fixation and inclusion, is prepared for the immunohistochemical exam first by de-paraffining the sections, then remoisturizing them, and finally submitting them to antigenic unmasking. The sample is then incubated with the primary antibody and then with by a biotine-streptovidina-kit detection system. To visualize the immunoreaction, Diaminobenzidine (DAB) is used as chromogen, which highlights the immunolabeling in brown. Densimetric and morphometric analyses of Fb-3 are obtained through optical microscope and image analysis software reading, in order to assess density in pixels (% of unit density) and the percentage of pixel immuno-labeled areas of the above quoted protein (see **Figure 2**) [2].

#### **4.2 Assessment of Fb-3 from serum, plasma and other biological liquids**

ELISA stands for Enzyme Linked Immuno Sorbent Assay. It is an immunological analysis technique used to assess any evidence of a particular antigen in a sample.

ELISA combines the specificity of the antigen–antibody reaction (immunological reaction) with the sensibility of a simple enzyme spectrophotometric dosage (see **Figure 3**).

Such technique is based on the assumption that, with adequate procedures, it is possible to conjugate the antibodies of a serum with some enzymes (peroxidase, alcalin phosphatase, beta-galactosidase) without altering their property to combine with the correspondent antigens. The enzyme used can catalyze a reaction on a suitable substratum with the formation of a colored terminal product, which allows highlighting the quantity of the antigen. In commercial formats, reactions are usually carried out inside polyvinyl or polystyrene wells (12 strip microplates with 8 wells each for a total of 96 wells) on which specific antibodies are attached for the antigen of interest or the antigen itself. The samples to analyze (plasma, serum, pleural liquid, broncho-aspirate, bronchoalveolar lavage, etc.) as well as reagents with interspersed

*Fibulin-3 as a Biomarker of Pleuric Involvement by Exposure to Fibers DOI: http://dx.doi.org/10.5772/intechopen.104448*

#### **Figure 2.**

*Figure. IHC determination of Fb-3 in lung tissues exposed to fluoro-edenite (FE). A–F: Sections of exposed lung tissue in which Fb-3 immunoexpression was detected in intraparenchymal stroma around bronchioles, bronchiolar epithelium, interstitium between alveoli, alveolar epithelium, and macrophages. A1-F1: Image analysis by software in which an evident both high (red color) and low (green color) immunostaining was detected in exposed lung. A-F, original magnification 20x; scale bar: 100 μm.*

#### **Figure 3.**

*Schematic drawing showing the antigen–antibody reaction (immunological reaction).*

lavages needed to remove any excess are incubated inside these wells. Lastly, the substratum is added, which generates the colored product.

Positivity is assessed analyzing occurrence or not of the color, following the reaction catalyzed by the enzyme on the substratum. Immunoenzymatic technique can be used for researching both antigens and antibodies and lends itself to several variations for numerous applications likewise.
