As expected, the specificity of the assay depends on using an appropriate pair of antobodies. The pair of antibodies works in conjunction, meaning that specificity and efficiency is limited by the least specific and efficient antibody. Therefore, excellent antibody quality is an important parameter for this method. We strongly recommend validating antibody specificity and efficiency. Validation of antibodies by immunofluorescence is also highly recommended. We have also validated pairs of antibodies by PLA. PLA can be performed in a single recognition experiment using different primary antibodies against the same protein. The couple showing the smaller number of dots is supposed to contain at least one limiting antibody that may be avoided for PLA assay.