The cells/sections on coverslips or the sections on grids were washed with distilled water before reverse transcription. The following components were used in the reaction mixture (RM) during testing: 1 × AMV reverse transcriptase buffer (Promega, 5 × AMV reverse transcriptase buffer: 250 mM Tris–HCl, 250 mM KCl, 50 mM MgCl2, 2.5 mM spermidine, 50 mM DTT), 0.2 U/µl AMV reverse transcriptase (Promega), 0.4 U/µl RNasin (Promega), 0.25 mM dATP, dGTP, dCTP and dTTP (Promega), 0.25 mM 5-bromo-2′-deoxyuridine triphosphate (BrdUTP, Sigma Aldrich), 0.05 mM biotin-16-2′-deoxyuridine-5′-triphosphate (biotin-dUTP, Roche), 0.05 mM digoxigenin-11-2′-deoxyuridine-5′-triphosphate (digoxigenin-dUTP, Roche), 0.05 mM Alexa Fluor® 555-aha-2′-deoxyuridine-5′-triphosphate (alexa-dUTP, Invitrogen), 0.05 mM ChromaTide® fluorescein-12-2′-deoxyuridine-5′-triphosphate (fluorescein-dUTP, Invitrogen) and 0.01 µg/µl oligonucleotides. The following oligonucleotides were used: oligonucleotides consisting of 15, 20 or 25 deoxythymidines (oligo dT15, Promega; oligo dT20, oligo dT25, Generi Biotech), oligonucleotides consisting of 15 deoxyadenines (oligo dA15, Generi Biotech) or a random hexanucleotide (Promega). The coverslips/grids were incubated on the 20 µl/10 µl drops of the RM in the moisture chamber for 1 h at 42°C and washed in 1 × PBS. Coverslips with cells labelled with alexa–dUTP or fluorescein-dUTP exclusively were washed in 1 × PBS and distilled water and mounted in Mowiol. The coverslips/grids with fluorescently non-labelled nucleotide analogues were incubated for 1 h with the primary antibody and after washing in PBS they were incubated for 1 h with the secondary antibody. The following primary antibodies were used in the comparative studies focused on the detection of cDNA tagged by bromodeoxyuridine (BrdU) or biotin-dUTP: a rabbit anti-biotin antibody (Enzo Lifesciences) diluted 1:100 in 1× PBS and a mouse anti-BrdU antibody (Roche) diluted 1:20 in 1× PBS. Digoxigenin-dUTP was detected by a mouse anti-digoxigenin antibody (Roche) diluted 1:100 in 1× PBS. For a comparison of the different antibodies against the BrdU, a rat anti-BrdU antibody (Abcam) diluted 1:100 in 1× PBS and a mouse anti-BrdU antibody (Becton-Dickinson) diluted 1:4 in 1× PBS were used in addition to the mouse anti-BrdU antibody purchased from Roche. For the detection of the splicing factor SC35, the mouse anti-SC35 antibody (Abcam) diluted 1:500 in 1× PBS was used. For LM, we used the anti-rabbit, anti-rat or anti-mouse secondary antibody conjugated with Cy3 or FITC fluorochrome. All of the secondary antibodies were diluted 1:100 in 1× PBS and were purchased from Jackson Immunoresearch. In the comparative studies focused on the detection of cDNA tagged by BrdU or biotin-dUTP and the comparision of different antibodies against BrdU, secondary antibodies conjugated with Cy3 were used exclusively. The chosen secondary antibodies (cat. number 115-165-146 and 111-165-144) provided a similar strength of the replication signal after the hypotonic introduction (10 (link)) of biotin-dUTP and BrdUTP into cells. For EM, we used an anti-mouse antibody conjugated with 10 nm of gold adduct diluted 1:100 in 1× PBS (Aurion). After incubation with the secondary antibody, the cells on coverslips were washed on drops of 1× PBS, then on drops of distilled water and then mounted in Mowiol. The sections on coverslips were washed on drops of 1× PBS, drops of distilled water, stained with DAPI for 20 min, washed in distilled water and mounted in Mowiol. The sections on grids were washed in 1× PBS, then in distilled water and subsequently incubated on drops of 3% uranyl acetate for 45 min. After that, the grids were washed in distilled water and dried.
The following components were used in the RM during the control experiments with DNA polymerase I, E. coli (Fermentas): 1 × DNA polymerase I, E. coli buffer (Fermentas, 10x polymerase I buffer: 500 mM Tris–HCl, 100 mM MgCl2, 10 mM DTT), 0.2 U/µl DNA polymerase I, E. coli, 0.25 mM dATP, dGTP, dCTP and dTTP (Promega), 0.25 mM BrdUTP and 0.05 mM biotin-dUTP.
In the other control experiments, the cells/sections were treated by DNase I or RNase A. We used DNase I (RNase-free, Fermentas, 0.1 U/µl in 10 mM Tris–Cl, pH 7.5, 2.5 mM MgCl2 and 0.1 mM CaCl2) or RNase A (DNase and protease free, Fermentas, 5 µg/ml in 10 mM Tris-Cl, pH 7.4).
The following components were used in the RM during the control experiments with DNA polymerase I, E. coli (Fermentas): 1 × DNA polymerase I, E. coli buffer (Fermentas, 10x polymerase I buffer: 500 mM Tris–HCl, 100 mM MgCl2, 10 mM DTT), 0.2 U/µl DNA polymerase I, E. coli, 0.25 mM dATP, dGTP, dCTP and dTTP (Promega), 0.25 mM BrdUTP and 0.05 mM biotin-dUTP.
In the other control experiments, the cells/sections were treated by DNase I or RNase A. We used DNase I (RNase-free, Fermentas, 0.1 U/µl in 10 mM Tris–Cl, pH 7.5, 2.5 mM MgCl2 and 0.1 mM CaCl2) or RNase A (DNase and protease free, Fermentas, 5 µg/ml in 10 mM Tris-Cl, pH 7.4).
