Samples of equine sarcoid (N°9) were derived from the archives of the Department of Pathology and Animal Health, University of Naples "Federico II". Normal skin from healthy horses was also examined. Formalin-fixed paraffin embedded tissue was available from each case. Sections taken from these blocks and stained by Haematoxylin and eosin (HE) were re-evaluated to confirm the diagnosis. The 9 samples were derived from 9 different animals. All sarcoids were known to be positive for BPV DNA .
Sarcoids and normal skin, from the same animals, were also collected and immediately frozen at -80°C for biochemical analysis.
E-DERM fibroblast cell lines, derived from horse dermis, were obtained from the American Type Culture Collection. EqSO1a and EqSO4b sarcoid derived cell lines have been previously described . All cells were maintained in culture in DMEM (Dulbecco's modified eagle medium) supplemented with 10% FBS (Gibco) in a 37°C humidified atmosphere of 5% CO2 in air.
Nine sarcoid samples and one normal skin sample were stained. Briefly, paraffin sections were deparaffinized, and blocked for endogenous peroxidase in 0.3% H2O2 in methanol for 20 min. Antigen enhancement was performed by pretreating with microwave heating (twice for 5 min each at 525 W). The anti-FHIT antibody (Santa Cruz, USA) was applied at 1:100 dilution overnight at room temperature in a humified chamber. The slides were washed three times with phosphate-buffered saline (PBS), then incubated with appropriate secondary antobodies as previously reported . Sections were washed three times with PBS and then incubated with streptavidin-conjugated to horseradish peroxidase (LSAB Kit; DakoCytomation, Denmark). Colour development was obtained by treatment with diaminobenzidine (DakoCytomation, Denmark) for 5 min. Sections were counterstained with Mayer's haematoxylin. In the corresponding negative control section, the primary antibodies were either omitted or replaced with appropriate normal sera.
The scoring of the immunoreactivity was determined in a 'blind' study by two observers (GB and AC). The intensity of labelling in each specimen was scored from absent to very strong immunosignal.
Immunofluorescence and confocal laser-scanning microscopy
E-DERM, EqSO1a and EqSO4b cell lines were grown for 2 days on coverslips, washed with PBS, fixed in 4% paraformaldehyde for 20 min, permeabilized with 0,1% triton X-100 in PBS 5 min. The slides were blocked with 2% BSA for 30 min. The anti-FHIT primary antibody was applied O/N at 4°C in a humified chamber at 1:50 dilution and after washing with PBS, incubated with Alexa Fluor 546 goat anti-rabbit 30 min at RT (Molecular Probes. Leiden, The Netherlands).
Finally, after washing with PBS, the slides were mounted in aqueous medium PBS:Glycerol 1:1 (Sigma, Milan, Italy). For scanning and photography, a confocal laser-scanning microscope LSM-510 (Zeiss, Gottingen, Germany) was used. Alexa Fluor 546 was irradiated at 543 nm and detected with a 560 nm bandpass filter.
Protein extraction and SDS PAGE/Western blotting
Three fresh tumour (S1, S2 and S3) and one sample of normal skin (N) were available for molecular analysis. These were snap frozen in liquid nitrogen and homogenized in ice-cold lysis buffer (50 mM Tris pH7.5; 150 mM NaCl; 1 mM EDTA; 0.25% Deoxicolic acid, 1% Triton X100) added with 20 mM sodium pyrophosphate, 0.1 mg/ml aprotinin, 2 mM phenylmethylsulphony fluoride (PMSF), 10 mM sodium orthovanadate (Na2VO3), and 50 mM NaF.
Cell lines were grown for 2 days in 60-mm dishes, washed with ice-cold phosphate saline buffer two times and lysed for 20 minutes in ice-cold lysis buffer. Tissue homogenates and cell lysates were clarified by centrifugation, and the quantity of proteins was determined by use of a protein assay kit (Bio-Rad Laboratories, Milan, Italy). 50 μg of total protein were boiled and fractionated in 15% SDS-PAGE gel. The proteins were blotted from the gel onto nitrocellulose membranes. The membranes were blocked with 5% non-fat dry milk in TBS buffer at room temperature, washed with TBS-0.1% Tween and incubated with anti-FHIT antibody at dilution 1: 500 (Santa Cruz). After appropriate washing steps, peroxidase-conjugated anti-rabbit IgG (Amersham Pharmacia Biotech) were applied for 1 hour at 1:5,000. After washing, bound antibody was visualized on ECL film (Amersham Pharmacia Biotech). The blots were stripped and reprobed against mouse anti-actin antibody (Calbiochem) at 1:5,000 to confirm equal loading of proteins in each lane.
Cell lines DNA and RNA extraction
Genomic DNA was extracted from one 60-mm dish respectively of E-DERM, EqSO1a and EqSO4b cell lines according to the Wizard Genomic DNA Purification Kit protocol (Promega Corporation, USA). The purified DNA was resuspended in TE.
Total RNA was extracted from E-DERM, EqSO1a and EqSO4b cell lines. The cell pellet derived from two 60-mm dishes, cultured as described above, was resuspended in 1 ml of Trizol (Invitrogen, Carlsbad, CA, USA). The RNA purification was obtained according to the Trizol protocol.
PCR and RT-PCR analyses
The equine specific FHIT CpG island was obtained by genomic DNA PCR amplification with the following primers: FHITCpGF TCCTGAGAGGGACAGTGGTT, FHITCpGR GGGGAGGGTTAGGGTGAG. The purified PCR product (573 bp) was directly sequenced by the dye terminator method (PRIMM Facility, Naples, Italy).
For all cell lines, cDNA was prepared, after DNAaseI RNAase free digestion, using 1 μg of total RNA according to the protocol of Quantitect Reverse Transcription for RT-PCR (Qiagen, Germany). The final volume was 20 μl. cDNA quality was tested through the amplification of the housekeeping gene GAPDH. The PCR primer sequences were GAPDHF GCCATCACCATCTTCCAG and GAPDHR GTTCACGCCCATCACAAAC and the PCR product lenght was 192 bp. The FHIT specific primers used were the following: FHITF2 GTCGGGAATTGTAGTCCTC, FHITF3 CTCTCTTCCCGGGTCTGTAA and FHITR2 GTTCACGAGGGCAAAGGATA. The F2 R2 product lenght was 359 bp; the F3 R3 product lenght was 451 bp. The purified PCR products were directly sequenced by the dye terminator method (PRIMM Facility, Naples, Italy).
DNA extraction from paraffin-embedded sarcoid samples, bisulfite conversion and sequencing
Genomic DNA was isolated from paraffin-embedded sections using standard procedures and was precipitated with 250 mM NaCl and isopropanol. Extracted DNA (0.25 - 1 μg) was bisulfite-converted using the EpiTect Bisulfite Kit (Qiagen, Germany), according to the protocol relative to the paraffin embedded samples. Samples were eluted in a final volume of 20 μl. To study the differential methylation status of FHIT putative promoter, bisulfite sequencing was performed. Briefly, 1 μl of bisulfite-converted DNA was amplified using a double step PCR (semi-nested PCR) carried out using Gradient PCR Express (Hybaid, Middlesex, UK). Step 1 primer sequences are: bisFHITF1 TTTTTTGAGAGGGATAGTGGTTTT and bisFHITR1 ACCAATACTCAAAAATAAACCCTCA; step 2 primer sequences are: bisFHITF2 GTTATTATGGTTTTTGATTGGGTTG and the above cited bisFHITR1. The second amplification was carried out using 1 μl of first amplification product as template. The thermal cycling conditions were: 95°C for 3 min, followed by 95°C for 30 sec, 52°C for 45 sec and 72°C for 45 sec for 25 (first amplification) or 35 (second amplification) cycles. After amplification, PCR products were recovered from agarose gel using QIAquick Gel Extraction Kit (Qiagen), following manufacturer's instructions. Purified PCR products were cloned in the pGEMeasy cloning vector (Promega Corporation, USA). After cloning, DNA fragments were sequenced by the dye terminator method (PRIMM Facility, Naples, Italy).