Supplementary Figure 2: Preservation of endogenous proteins and their probe-binding affinities in SHIELD tissue.

(a) Venn diagram of antigenicity test results. All 53 tested antibodies compatible with PFA-fixed control tissue worked in SHIELD tissue. (b) Protein loss assay in the high-temperature tissue clearing condition (200 mM SDS, 70°C, 12 hr). N = 3 tissues. (c, d) FP preservation in SHIELD-processed tissues after multiple rounds of staining and destaining. (c) Endogenous YFP signal retention of PFA and SHIELD tissues after destaining treatments corresponding to 5 rounds of stainings. N=4 tissues. (d) Multiround staining images from the hippocampal region. Break lines indicate destaining steps. For the second round, secondary antibodies were added to confirm complete unbinding of the primary antibodies imaged at the first round. Thy1-H+ YFP mouse SHIELD tissue was used. Scale bars = 100 μm (e) Representative 21 immunofluorescence images in PFA, GA, and SHIELD-processed tissues. Scale bar = 100 μm. The same imaging and display settings were used for each antibody. (f) The contrast-adjusted reproduction of Figure 2i. The selected images were adjusted for better visual comparison. Scale bar = 20 μm (g) Representative images comparing the immunofluorescence of MBP and NF-H in uncleared PFA, GA, and SHIELD tissues. To exclude the effect of tissue clearing on antigenicity, uncleared sections were used. Scale bar = 20 μm. MBP, myelin basic protein; NF-H, neurofilament-H. (h) SHIELD maintained a SNR of MBP and NF-H immunofluorescences similar to that of the PFA-control in immunohistochemistry, indicating minimal epitope damage by P3PE crosslinking. N = 4 tissues. Unpaired T-test, *P < 0.05. Mean +/- standard error mean was used for all the bar graphs.