Extended Data Fig. 4: Strategies for reducing intravascular precipitates during ThICK staining.
From: Antibody stabilization for thermally accelerated deep immunostaining
a, Optimization of ThICK-staining protocol. Additives were added in various incubation steps while vascular precipitation of SPEARs was globally quantified for each imaged tissue stack and normalized against the imaged tissue volume (see Methods). The optimization was performed iteratively for four rounds (grouped in colors). For all experiments, permeabilization was performed at 37 °C for 1 day, ThICK staining was performed at 55 °C for 16 hours, and post-washing was performed at room temperature for 1 day. Abbreviations: BSA, bovine serum albumin; GnCl, guanidinium chloride; PBST, 1× phosphate-buffered saline with 0.3% v/v Triton X-100; TMAO, trimethylamine oxide; Tx, Triton X-100; SDC, sodium deoxycholate; SDS, sodium dodecyl sulfate. b, and c, Post-imaging removal of intravascular SPEAR precipitates. b, Approach for segmenting and removing intravascular SPEAR precipitates using a commercial software (Imaris, see Methods). c, Removal of vasoactive intestinal peptide SPEAR intravascular precipitates after one round of image processing.
