Cloning the S100A7 gene
The open reading frame of the human S100A7 gene was obtained by PCR using a cDNA template from tracheal tumor tissue ("Projeto Genoma Clínico", FAPESP and Ludwig Institute for Cancer Research, Brazil) [13, 14]. Primers containing cleavage sites for the restriction enzyme Xho I (5' ATGGATCCCTCGAG ATGAGCAACACTCAAG 3') and Hind III (5' ATAAGCTT TCACTGGCTGCCCCCGGAA 3') were synthesized by Invitrogen (Carlsbad, CA, USA). For PCR, 10 μg from trachea tissue furnished by the human tissue cDNA bank of the Regional Center for Hemotherapy of Ribeirão Preto, University of São Paulo, was used. The reaction mixture contained 1 mM dNTPs, 7.5 mM sense and complementary primers, 1 U Platinum Taq DNA polymerase in 50 mM Tris-HCl, pH 8.8, containing 500 mM KCl, 5 mM MgCl2 (Invitrogen) and 1% Tween 20 (Sigma, Saint Louis, MO, USA). PCR consisted of an initial denaturation step of 5 min at 94°C, followed by 35 cycles of 40 s at 94°C (denaturation), 1 min of annealing at 53°C (the specific annealing temperature for the S100A7 gene) and 1 min at 72°C (extension). A second nested PCR assay was then performed using 1 μl of the reaction mixture described above and annealing at 60°C. The amplified 306 bp product was purified on 1.0% (w/v) agarose gel using the Wizard SV Gel, PCR Clean-Up System kit (Promega, Madison, WI, USA) and inserted into the propagation vector pCR2.1-TOPO using the TOPO-TA cloning kit (Invitrogen) according to the manufacturer's instructions. The construct was introduced into E. coli DH5α by the thermal shock method described by Sambrook and Russell  and the recombinants were selected on solid Luria-Bertani (LB) medium containing ampicillin (100 μg/ml). Plasmid DNA was extracted with the plasmidPrep Mini Spin kit (GE Healthcare, Little Chalfont, Buckinghamshire, UK) according to the manufacturer's instructions. The structure of the construct was confirmed by nucleotide sequencing using the Thermocycle Sequencing BigDye Terminator kit (Applied Biosystems, Carlsbad, CA, USA) in an ABI Prism 377 automatic sequencer (Applied Biosystems). The S100A7 fragment was removed from the pCR2.1-TOPO vector by digestion with the restriction enzymes Xho I and Hind III and attached to the expression vector pAE, which contained the 6xHis-tag peptide in the N-terminal region , according to the protocol for the enzyme T4 DNA ligase (Promega). The construct was introduced into competent E. coli DH5α cells with the Subcloning Efficiency™ Chemically Competent E. coli kit (Invitrogen). The transformants were selected by PCR and plasmid DNA was extracted as described by Sambrook and Russell  and sequenced using the DYEnamic ET Dye Terminator Sequencing kit (GE Healthcare) with the automatic sequencer MegaBace™ 1000 (GE Healthcare). This indicated that the insertion of the 306 bp fragment corresponding to the S100A7 gene into the expression vector pAE was correct. The sequence of the fragment was identical to that deposited in GenBank as accession number 002963.
Expression of rS100A7 (His-tag) protein
The E. coli BL21::DE3 host was transformed with the vector pAE+S100A7 and plated onto selective medium. A single colony was used for the inoculation of 200 ml CircleGrow liquid medium containing 100 μg/mL ampicillin into a 500 ml Erlenmeyer flask. The culture was maintained at 37°C and stirred until it reached an absorbance of approximately 0.5 at 600 nm. Isopropyl-β-D-1-thiolgalactopyranoside (IPTG) at 1 or 10 mM was added to try to increase the amount of protein produced.
Purification of rS100A7 (His-tag) protein
E. coli BL21::DE3 cultivated in 500 ml of culture medium was centrifuged at 5,000 g for 10 min and the pellets were stored at -80°C. They were resuspended in 5 ml lysis buffer A (6 M GuHCl; 0.1 M NaH2PO4, 10 mM Tris-Cl; pH 8.0) and maintained at 4°C for one hour with shaking at 1,000 rpm. The lysate was centrifuged at 10,000 g for 30 min at 4°C (Eppendorf 5804 R) and 1.0 ml the supernatant was loaded onto an Ni-NTA SuperFlow affinity chromatography minicolumn equilibrated with buffer A (Qiagen, Hilden, Düsseldorf, Germany). The remainder of the lysate (4.0 ml) was processed at 1.0 ml/minicolumn. Each was centrifuged at 700 g for 2 min. After loading the minicolumn was washed with 600 μl buffer C (8 M urea; 0.1 M NaH2PO4, 10.0 mM Tris-Cl; pH 6.3) and centrifuged at 700 g for 2 min two times. The protein was eluted with 200 μl Buffer E (8 M urea; 0.1 M NaH2PO4, 10.0 mM Tris-Cl; pH 4.5) and centrifuged at 700 g for 2 min, three times.
The homogeneity of the recombinant S100A7 (His-tag) protein (10 μg) was evaluated by 12.5% SDS-PAGE . The gel was stained with colloidal Coomassie blue G-250 (Serva, Heidelberg, Germany) according to Neuhoff et al. . The amount of recombinant protein was measured with a DC Protein Assay kit (Bio-Rad) based on the method of Bradford  and determined by absorbance at 595 nm in a Versa-Max microplate reader (Molecular Devices, Sunnyvale, CA, USA).
Characterization of the recombinant S100A7 (His-tag) by mass spectrometry
Recombinant S100A7 (His-tag) protein was characterized after in situ trypsin digestion of the SDS-PAGE gel band corresponding to 11.5 kDa. The band was cut from the gel, destained with 0.1 M NH4HCO3 in 50% acetonitrile, dehydrated with 100% acetonitrile, dried in a SpeedVac (Savant Inc., Ramsey, MN, USA), and rehydrated with 20 μl 0.1 M NH4HCO3 containing 0.5 μg trypsin (Promega). The protein was digested at 37°C for 24 h. The hydrolysate was desalted in a microtip filled with POROS 50 R2 resin (PerSeptive Biosystems, Foster City, CA, US) and analyzed with a MALDI-TOF/TOF mass spectrometer (Axima Performance, Kratos-Shimadzu Biotech, Manchester, UK) equipped with a collision chamber for collision-induced dissociation (CID), with helium as the collision gas. The desalted hydrolysate was dissolved in 10 μl matrix solution containing 5 mg/ml alpha-cyano-4-hydroxycinnamic acid in 0.1% trifluoroacetic acid and 50% acetonitrile. Two microliters of the sample-matrix solution were applied to the stainless steel MALDI plate and allowed to evaporate. The mass spectrometer was calibrated with a mixture of angiotensin II, ACTH fragment 18-39, bradykinin fragment 1-7 and the oxidized B chain of bovine insulin (Sigma). The PMF and MS/MS spectra were analyzed with Mascot software V.2.2.4 (Matrix Science, London, UK) and compared with the structure of S100A7 (His-tag) construct.
The molecular weight of the intact rS100A7 (His-tag) protein was determined with MALDI-TOF/TOF-MS operated in the linear mode after desalting with POROS 50 R2. The instrument was calibrated with insulin, cytochrome C, chymotrypsin and bovine serum albumin.
Production of polyclonal antibody anti-rS100A7
The anti-recombinant S100A7 (His-tag) rabbit serum was obtained according to standard protocols . Briefly, two adult female New Zealand white rabbits were immunized with 2.0 mg/animal of purified recombinant S100A7 (His-tag) protein emulsified in complete or incomplete Freund's adjuvant (Sigma) by subcutaneous or intramuscular injection, respectively. Before immunization, blood was obtained from each animal to prepare non-immune serum. The immunization efficiency was analyzed by Western blotting and serum samples were stored at -20°C.
Reaction of rabbit polyclonal antibody with recombinant S100A7 (His-tag) protein under denaturing and reducing conditions
Western blotting was performed after 200 ng of purified recombinant S100A7 (His-tag) protein was submitted to 12.5% SDS-PAGE  and the protein was transferred from the gel to a polyvinyldene fluoride (PVDF) membrane in buffer containing 25 mM Tris, 192 mM glycine, pH 8.3, and 10% methanol. A constant voltage of 35 V and 100 mA was applied. A PVDF membrane and the Chromogenic Western Blot Immunodetection kit (Invitrogen) were used according to manufacturer's instructions. The membranes were incubated separately with the primary antibodies, i.e., the anti-recombinant S100A7 (His-tag) rabbit serum at 1:5,000 dilution, and the positive control monoclonal antibody S100A7 (Novocastra, Newcastle Upon Tyne, UK) at 1:1,000 dilution. Finally, the membranes were washed in MilliQ water and incubated with the chromogenic substrate 5-bromo-4-chloro-3-indolyl-1-phosphatase (BCIP) and nitroblue tetrazolium (NBT). After protein labeling, the membranes were washed with MilliQ water in order to stop the reactions.
Interaction of the polyclonal antibody anti-rS100A7 with tissue microarray (TMA)
The anti-recombinant S100A7 (His-tag) rabbit serum was used for the detection of S100A7 by immunohistochemistry in tissue samples arranged in a TMA. Representative areas of tissues fixed in formalin and embedded in paraffin were selected randomly and used for the construction of a TMA block which contained up to four spots each of non-neoplastic tissue (Malpighian epithelium) and of neoplastic tissue (squamous cell carcinoma) from eight cases of neoplasias of the head and neck region (larynx). The TMA experiment was conducted using the polyclonal antibody at 1:2,000 to 1:16,000 dilutions. The anti-S100A7 monoclonal antibody (Novocastra) was used as a positive control at a dilution of 1:50 to 1:200. All dilutions were incubated at 4°C for 16 h after antigen recovery under the following conditions: incubation with 10 mM sodium citrate, pH 6.0, or incubation with 1 mM EDTA, pH 8.0, in a pressure cooker for 40 min, or in the absence of antigen recovery. The reaction was amplified using the NovoLink Polymer Detection System kit (Novocastra) according to manufacturer's instructions. The reaction was developed with the chromogenic substrate containing 0.10% diamine benzidine (Sigma, Saint Louis, MO, US), 0.06% hydrogen peroxide, and 1% dimethyl sulfoxide (Labsynth) in PBS buffer. Sections containing TMA were then counterstained with Harris hematoxylin and rapidly immersed in 0.5% ammonium hydroxide. Immunoreactivity was scored as follows: absent (no color) or positive (brownish red) present in up to 5% of the cells of interest (+1), present in > 5% and ≤ 25% of the cells of interest (+2), present in > 25% and ≤ 50% of the cells of interest (+3), and present in > 50% of the cells of interest (+4).
The controls were: positive - spots containing human lung - and the negative control was without the primary antibody.