On 2017 Aug 26, Giorgio Cattoretti commented:

The method published is based on blocking the detection of previously deposited antibodies by a second staining round via an undisclosed amount of monomeric Fab fragments of secondary antibodies. “No cross-reactivity was observed between secondary Abs targeting primary Abs from the same species or with the same isotype. The absence of cross-reactivity was dependent on incubation with a blocking buffer before each restaining step (fig. S1) “ [quoted from the paper]. But on page 4, quoted again, ”tissue section was repeatedly stained, destained, and restained with the same anti-CD3 Ab" (legend to Fig. S5), in order to demonstrate "the absence of steric hindrance ….. upon successful consecutive cycles of staining/bleaching using the same marker (fig. S5)." If the block was applied after each staining round in both cases, no staining at all should be the expected result, as the Authors have shown in Fig. S1. If instead no block was applied, resulting in crossreactions between previous staining layers and the successive, any claim regarding steric hindrance has a weak scientific basis at best. In either scenario, the contradiction question the validity of the method, unless explained. In addition, a “dehydration” step in xylene (Fig. 1) most likely entails a reduction of tissue antigenicity, thus reproducibility, as we published (PMID: 26487185) six months before this paper was submitted and afterwards (PMID: 28692376 ): this topic is not sufficiently addressed.

On 2017 Aug 26, Giorgio Cattoretti commented:

The method published is based on blocking the detection of previously deposited antibodies by a second staining round via an undisclosed amount of monomeric Fab fragments of secondary antibodies. “No cross-reactivity was observed between secondary Abs targeting primary Abs from the same species or with the same isotype. The absence of cross-reactivity was dependent on incubation with a blocking buffer before each restaining step (fig. S1) “ [quoted from the paper]. But on page 4, quoted again, ”tissue section was repeatedly stained, destained, and restained with the same anti-CD3 Ab" (legend to Fig. S5), in order to demonstrate "the absence of steric hindrance ….. upon successful consecutive cycles of staining/bleaching using the same marker (fig. S5)." If the block was applied after each staining round in both cases, no staining at all should be the expected result, as the Authors have shown in Fig. S1. If instead no block was applied, resulting in crossreactions between previous staining layers and the successive, any claim regarding steric hindrance has a weak scientific basis at best. In either scenario, the contradiction question the validity of the method, unless explained. In addition, a “dehydration” step in xylene (Fig. 1) most likely entails a reduction of tissue antigenicity, thus reproducibility, as we published (PMID: 26487185) six months before this paper was submitted and afterwards (PMID: 28692376 ): this topic is not sufficiently addressed.