Table 1.

Flowchart of cyclic antibody staining and removal with 2ME/SDS and GnHCl.

2ME/SDS staining and stripping method
1	Perform ARx on dewaxed sections affixed to positively charged glass slides
2	Allow to cool to about 50C or lower
3	Acquire the AF image for all the channels deemed necessary (optional)
4	Perform the first IF stain in 100 mM trehalose-containing dilution buffer
5	Mount with 60% Glycerol in PBS, 0.2% N-propyl Gallate, and 584 mM sucrose mounting medium containing 5.45 µM DAPI
6	Label the slide, acquire the images for all channels, including DAPI and AF, if not acquired before
7	Unmount the slides in buffer/distilled water
8	Transfer to Tris buffer
9	Immerse for 30 min in preheated (56C) 2ME/SDS buffer with agitation
10	Transfer to Tris buffer and wash extensively with TBS-Ts buffer
11	Repeat from step 4 with additional positive and/or negative antibodies
12	Store in 50% glycerol at −20C/−80C for extended storage, before returning to step 4 or proceed as below
13	Perform H&E or insoluble stainings if final
GnHCl staining and stripping method
1	Perform ARx on dewaxed sections affixed to positively charged glass slides
2	Allow to cool to about 50C or lower
3	Acquire the AF image for all the channels deemed necessary (optional)
4	Perform the first IF stain in 100 mM trehalose-containing dilution buffer
5	Mount with 60% Glycerol in PBS, 0.2% N-propyl Gallate, and 584 mM sucrose mounting medium containing 5.45 µM DAPI
6	Label the slide, acquire the images for all channels, including DAPI and AF, if not acquired before
7	Unmount the slides in buffer/distilled water
8	Transfer to Tris buffer
9	Immerse in GnHCl 6 M at 40C for 10 min
10	Transfer to GnHCl 6 M + 290 mM sucrose at 40C for 10 min
11	Transfer to 6 M urea + 290 mM sucrose at 40C for 10 min
12	Transfer to 3 M urea + 290 mM sucrose at 40C for 10 min
13	Transfer to Tris buffer and wash with TBS-Ts buffer
14	Transfer to 10 mM EDTA AR buffer pH 8, boil for 1 min, cool to 50C
15	Transfer to TBS-Ts buffer
16	Repeat from step 4 with additional positive and/or negative antibodies
17	Store in 50% glycerol at −20C/−80C for extended storage, before returning to step 4 or proceed as below
18	Perform H&E or insoluble stainings, if final

The sequential steps for repeated antibody staining and removal with 2ME/SDS and GnHCl are listed. Abbreviations: 2ME/SDS, 2-mercaptoethanol/sodium dodecyl sulfate stripping buffer; GnHCl, guanidinium hydrochloride; ARx, two-step antigen retrieval method³⁰; AF, autofluorescence; IF, immunofluorescence; PBS, phosphate-buffered saline; TBS-Ts, Tris-buffered saline containing Tween-20 and sucrose; DAPI, 4′6-diamidino-2-phenylindole, dihydrochloride; H&E, hematoxylin and eosin. For detailed composition of buffers and solutions, see text.