The cells were washed using Olink buffer A and incubated in amplification-polymerase solution, containing oligonucleotides probes with red colour fluorophores and polymerase, at 37°C in a humidity chamber for 100 minutes for rolling cycle amplification.
The cells were again subjected to a 10 minute wash and a 5 minute wash in Buffer A. The ligation reaction was carried out at 37°C for one hour in a humid chamber followed by a 10 and 5 minute wash in Buffer A. The cells were then incubated with the amplification-polymerase solution for two hours at 37°C in a darkened humidified chamber.
The slides were incubated in amplification-polymerase solution for 100 min at 37°C and then washed in wash buffer B. To co-stain CD8, rat anti-CD8 antibody was added in the antibody diluent with primary antibodies for PLA and the slide were incubated with Alexa Fluor 488 conjugated anti-rat IgG (Abcam) after washing in wash buffer B. Nucleus was stained with DAPI.
The nested PCR was performed with the Qiagen Taq DNA Polymerase and solution Q (Qiagen) on a PTC100 cycler (Biozym, Germany).
For the PCR amplification of the 3' variable region of the cagA gene that contains the EPIYA sequences, 20 to 100 ng of DNA were added to 1 % Taq DNA polymerase buffer solution (KCl 50 mM and Tris-HCl 10 mM), 1.5 mM MgCl2, 100 μM of each deoxynucleotide, 1.0 U Platinum Taq DNA polymerase (Invitrogen, São Paulo, Brazil), and 10 pmol of each primer, for a total solution volume of 20 μL.
For the PCR amplification of the 3' variable region of the cagA gene (that contains the EPIYA sequences), 20 to 100 ng of DNA were added to 1% Taq DNA polymerase buffer solution (KCl 50 mM and Tris HCl 10 mM, pH, 8.0), 1.5 mM MgCl2, 100 μM of each deoxynucleotide, 1.0 U Platinum Taq DNA polymerase (Invitrogen, São Paulo, Brazil), and 10 pmol of each primer, for a total solution volume of 20 μL.
The secondary PCR was performed with 5 μL of primary PCR and 45 μL of polymerase chain reaction solution.
PCR amplification was performed using Taq polymerase and Q solution according to the manufacturer's instructions (Qiagen, Valencia, CA).
Advantage 2 PCR kit (Clontech, Inc., CA, USA) was used as polymerase chain reaction solution, which contains 1x PCR buffer 40 mM Tricine-KOH (pH 8.7), 15 mM KOAc, 3.5 mM Mg OAc 2, 3.75 μg/mL BSA, Tween 20 0.005%, 25 mM dNTP, 12.5 μM primer, and 1x Advantage 2 polymerase mix.
They suggested that these loci be split into two (or more, if necessary) loci by designing internal primers to increase DNA polymerase fidelity; this solution would also remove issues with size homoplasy by insuring that all variation is produced by a single variable region.
