On 2017 Nov 17, Giorgio Cattoretti commented:

In 2014 [PMID: 24794148] we demonstrated that a pH2 Glycine buffer and a boiling 6M Urea buffer fail to remove primary antibodies in an exquisitely affinity-dependent fashion. Secondary antibodies increase the primary antibody affinity, further preventing the antibody complex removal with those buffers. Antibodies were not "degraded " to the point of not being recognizable by a second step, as shown in our paper. Later this year [PMID: 28692376 ] we published two separate and efficient antibody removal technique which preserve tissue antigenicity entirely over ten cycles and more. The method allows whole slide immunostaining with routine reagents and open source analysis tools as per this manuscript, but with far more markers per slide (50 or more). This manuscript describes a partially efficient two-step method for “detachment” but little rationale for the choice of buffers and none of the multiple previous references dealing with antibody removal (see our 2014 manuscript for a list). The fact that the Authors reproduce our previous observations is comforting; unfortunately misses the scientific rationale we provided and the solution of the problem we published.

On 2017 Nov 17, Giorgio Cattoretti commented:

In 2014 [PMID: 24794148] we demonstrated that a pH2 Glycine buffer and a boiling 6M Urea buffer fail to remove primary antibodies in an exquisitely affinity-dependent fashion. Secondary antibodies increase the primary antibody affinity, further preventing the antibody complex removal with those buffers. Antibodies were not "degraded " to the point of not being recognizable by a second step, as shown in our paper. Later this year [PMID: 28692376 ] we published two separate and efficient antibody removal technique which preserve tissue antigenicity entirely over ten cycles and more. The method allows whole slide immunostaining with routine reagents and open source analysis tools as per this manuscript, but with far more markers per slide (50 or more). This manuscript describes a partially efficient two-step method for “detachment” but little rationale for the choice of buffers and none of the multiple previous references dealing with antibody removal (see our 2014 manuscript for a list). The fact that the Authors reproduce our previous observations is comforting; unfortunately misses the scientific rationale we provided and the solution of the problem we published.