The amino acid sequence of human nestin was carefully analyzed and compared to all other species. A 12-mer peptide of PEVGDEEALRPL from human nestin corresponding to amino acids 681–692 (NM_ 006617) was selected as immunogen. A cysteine residue was added to the C-terminus end of the peptide to facilitate the conjugation to carrier protein. Immunograde peptide was purchased from Thermo Electron Corporation, GmbH, Ulm, Germany. Keyhole Limpet Hemocyanin (KLH)-conjugated peptide was used for generating mouse monoclonal antibody according to standard protocol with minor modifications (11 , 12 ).

Briefly, 5 mg of Keyhole Limpet Hemo-cyanin (KLH) (Sigma, cat. no: H7017) was dissolved in 1 ml of deionized water. One mg of Maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) dissolved in 200 µl of Dimethyl Formamide (DMF) was then added to the carrier protein solution. The mixture was incubated at room temperature for 2 hrs with gentle stirring followed by dialysis against large volume of Phosphate Buffer Saline (PBS) overnight. In a separate tube 5 mg of peptide was dissolved in 1 ml of Phosphate Buffered Saline (PBS). MBS-activated protein was then added to the peptide solution and the mixture was incubated for 4 hrs at room temperature. After overnight dialysis against PBS, the conjugate was stored at −20 °C for later use. All chemicals for bioconjugation were purchased from Sigma, St. Louis, USA. The same procedure was performed for conjugation of peptide to BSA.

To check the efficiency of conjugation, 10 ug of peptide-BSA was mixed with 10 ul of sample buffer, boiled for 2 – 5 min and cooled on ice. Electerophoresis was performed in a 10% SDS-PAGE gel with a mini-PROTEAN electerophoresis instrument (Bio-Rad Laboratories, Hercules,CA,USA) 100 mA for 1 hr. The gel was stained with Coomassie Blue R-250 (Sigma). The change in mobility shift of conjugated BSA represented the efficiency of conjugation.

Four BALB/c mice were used for peptide immunization. Each mouse was immunized 5 times with an interval of two weeks. The first immunization was performed using Freund's complete adjuvant. Incomplete Freund's adjuvant was used for the 2^nd, 3^rd, 4^th, and 5^th immunization.

For the first immunization, 100 ug of conjugated KLH-peptide was mixed with an equal volume of Freund's complete adjuvant and injected Intra Peritoneally (IP) not exceeding 100 ul total volume. For the subsequent immunizations 50 ug of peptide-KLH were injected with Freund's incomplete adjuvant. Three days before the cell fusion, 20 ug of KLH-peptide (without any adjuvant) was injected intravenously.

One week before the last immunization, mice were bled by a vertical incision of the tail vein. Serum ELISA assay was performed as follows:

Wells of ELISA plate (Nunc, Germany) were coated with 100 ul of the immunizing peptide (20 µg/ml in PBS) at 37 °C for one hr followed by overnight incubation at 4 °C. Next day the plate was washed 3 times with PBS containing 0.05% Tween 20 (PBS-T) for 5 min. The plate was blocked with 2.5% BSA at 37 °C for 1.5 hr. Wells were then washed 3 times as above and mice sera were added to the wells in two fold serial dilutions starting from 1:100. The plate was incubated at 37 °C for 1.5 hr and washed again with PBS-T.

At the next step, 100 µl of 1:1000 dilution of HRP-conjugated rabbit anti-mouse Ig (Avicenna Research Institute, Iran) was added to the wells and incubation was continued for 1.5 hr at 37 °C. After washing, 100 ul of Tetramethylbenzidine (TMB) substrate was added to each well and the plate was incubated at room temperature in a dark place. After 15 min, the reaction was stopped by adding 30 ul of stopping solution (0.16 M H2SO4) to each well.

The Optical Density (OD) of the reactions was measured at 450 nm by an ELISA reader (BioTek, USA). Negative controls included omission of coating layer, serum (as primary antibody) or combination of both. Mice with higher titer of specific antibody were selected for fusion.

Mouse myeloma SP2/0 cell line was used as fusion partner. Cells were cultured in RPMI (GIBCO) and 10% FBS until reaching to >70% confluency. To collect mouse peritoneal macrophages, 5 ml of RPMI was injected into the peritoneal cavity of unimmunized BALB/c mouse with subsequent aspiration and collecting the peritoneal cells at sterile conditions (∼3x10⁶ cells). The collected peritoneal fluid was washed twice with RPMI. The cells were incubated in RPMI with 20% FBS for 24–48 hrs at 37 °C with 5% CO2. Spleen of the immunized mouse was removed at sterile conditions. To separate spleen cells, 10 ml of RPMI was injected to the spleen from different angles. The collected cells were washed twice with RPMI for 10 min and centrifuged at 1000 rpm.

A 50 ml sterile Falcon tube was selected and SP2/0 cells were mixed with the spleen cells at a ratio of 1:10 (1 SP2/0 and 10 spleen cells). Mixture was washed twice with RPMI. 800 ul of pre-warmed (37 °C) 50% PEG 1500 (Sigma) was added to the cell pellet slowly by mixing at the same time. Immediately after adding PEG, 20 ml of pre-warmed RPMI was added to the tube to dilute out PEG. Cells were centrifuged at 22 °C for 5 min and at 500 rpm. The cells were washed twice. HAT medium was added based on the number of spleen cells for fusion. Selective HAT medium was added to the pellet (2x10⁶ cells/ml) and cells were seeded into a 96-well plate. The cells were incubated at 37 °C with 5% CO2 for 2–3 days. Cell growth and colony formations were examined daily. Colonies appeared after 5–10 days. Once the colony diameter reached to 1 mm the presence of antibody against the immunized peptide was determined by ELISA. After two weeks of incubation 100 ul of supernatant from each well were separated and ELISA assay was performed using peptide alone, KLH-peptide, BSA-peptide, and KLH only as coating antigen.

The monoclonal antibodies were purified from culture supernatants by affinity chromatography based on its isotype. Briefly, culture supernatants were filtered through 45 µm filter and pH was adjusted to 7.5. Antibodies were affinity purified using a column of CNBr-activated sepharose 4B (GE Healthcare, Sweden) conjugated to immunogenic peptide. The elution was performed using 0.1 mol/l glycine pH=2.7. The pH of eluted antibody was adjusted to 7.0 with 1 mol/l Tris buffer pH=9.0. The eluted anti-bodies were dialyzed overnight against PBS pH=7.5 and the reactivity of the antibodies were measured by ELISA as described above.

Western blotting was performed to see pattern of reactivity of anti-nestin monoclonal antibodies with different isoforms of this protein according to the protocol we reported elsewhere with minor modification (12 ).

Briefly, a panel of cell lines with different origin known to express nestin (Table 1 ) (all from ATCC, USA), were cultured in their recommended medium, harvested and lysed in 1 ml of lysis buffer containing 1% Triton X-100, 50 mM Tris-HCl, pH=7.4, 150 mM NaCl, 5 mM EDTA, and 1% protease inhibitor cocktail (Sigma) for 1 hr on ice with a 15 min interval of vortexing for 30 sec. The protein concentration of lyaste was measured by Thermo Scientific BCA Protein Assay Kit according to the manufacturer's instructions (Thermo Scientific, IL, USA). Twenty µg of cell lysates were run under both reducing and non-reducing conditions on a 6% SDS-PAGE gel at 100 V for 2 hr. After electrophoresis, resolved proteins were transferred onto Immobilon-PVDF membranes (Millipore Corporation, USA). The membranes were blocked for overnight at +4 °C with 5% non-fat milk in PBS plus 0.05% Tween 20 (PBS-T). All immunostainings were performed in PBS-T supplemented with 3% non-fat milk. Filters were incubated with 3 ug of affinity-purified anti-nestin monoclonal antibody 1.5 hr at room temperature. After extensive washing with PBS-T the filters were incubated with peroxidase-conjugated sheep anti-mouse immunoglobulins (ACECR, Tehran, IRAN) for 1 hr at room temperature followed by washing and developing with ECL chemiluminescence detection system (GE Healthcare, Uppsala, Sweden).

Total RNA was extracted from cell lines and tissue samples using RNA-Bee reagent (BioSite, Täby, Sweden) according to the manufacturer's instruction. The quality of the RNA samples was determined by agarose gel electrophoresis after staining with ethidium bromide, and visualization under UV light. Total RNA was unfolded at 65 °C for 10 min. cDNA was then synthesized using 5 µg of total RNA in 20 µL reaction mixture consisting of 4 µL of 5x reaction buffer, 1 µL of 10 mM dNTPs, 1 µL of 10 pmol/mL random hexamers (N6), and 200 U M-MuLV reverse transcriptase (Fermentas GmbH, St. Leon-Rot, Germany). The reaction mixture was incubated at 42 °C for 45 min. The amplification was performed with 1 min denaturating at 95 °C followed by 35 cycles of 94, 52 and 72 °C, 1 min each with a 2.5 mM MgCl2, using AmpliTaq Gold DNA polymerase (Applied Biosystems, USA) using species-specific nestin primers (Table 2 ).

The cells were harvested using 0.5% trypsin and 0.1% EDTA (Gibco) loaded 1–2×10⁴ cells on 8 well laminated glass slide (Marienfeld, Germany) that homogenized in RPMI 1640 contain 20% FBS with subsequent incubation in moisturized conditions for overnight.

After overnight incubation the medium was removed and the cells were washed with TBS for three times (3×3 min). Slides were dried at room temperature for 15 min, acetone-fixed (at −20 °C), permeabilized for 2 min and kept at 4 °C for 30 min until slides were dried. Slides were washed with Tris-Buffered Saline, pH=7.4 containing 5% bovine serum albumin (TBS-Glycerol) three times (3×3min).

Slides were blocked with 5% sheep serum for 10 min at room temperature. The primary antibody Mab anti-nestin (4G10G8) diluted with TBS-BSA to a final concentration of 5 µg/mL, respectively, were incubated at room temperature for 60 min and then washed with TBS-BSA three times (3×3 min).

Fluorescein Isothiocyanate (FITC)-conjugated Sheep anti-mouse (ACECR, Tehran, Iran) was diluted with TBS-BSA in a ratio of 1:50 and incubated at room temperature for 45 min. Negative antibody control slides were incubated with mouse IgG1 at a final concentration of 10 µg/mL in TBS-BSA. After washing with TBS-BSA, the nuclei were counterstained by 4′,6-diamidino-2-phenylindole di-hydrochloride (DAPI) (Calbiochem, USA) at 1 µg/ml for 5 min, then the slides were washed, mounted in TBS-glycerol 80% and examined under a fluorescence microscope (Olympus, Tokyo, Japan).

Bovine sertoli cells were harvested by 0.5% trypsin and 0.1% EDTA (Gibco) and permeabilized by permeabilizing solution (Becton Dickinson, USA). According to the related protocol, sample analysis and data acquisition were performed by Flomax flow cytometry analysis software (Partec, Germany).