Currently, fluorescent imaging using secondary antibodies is single-color, meaning that only one fluorescent secondary antibody is selected. This step primarily aims to determine whether the TotalSeq™-A series primary antibodies have successfully bound to the antigens in the tissue while excluding any abnormalities caused by experimental operations during antibody incubation.

Sheared salmon sperm DNA is added during the preparation of blocking solution and antibody incubation solution to reduce non-specific adsorption of antibody-derived-tag (ADT) to the tissue.

Repeated freezing and thawing are not recommended. It is recommended to aliquot the original 4% PFA into smaller tubes after the first thaw and store them at -20°C for long-term storage until the expiration date. For short-term use within one week, it is recommended to store at 4°C.

（1）Commercial protein panels, such as TotalSeq™-A Mouse Universal Cocktail, V1.0 (Biolegend, Cat. No. 199901) and TotalSeq™-A Human Universal Cocktail, V1.0 (Biolegend, Cat. No. 399907). These panels can detect 119 and 154 proteins (excluding isotype controls), respectively. The proteins targeted by these panels are mainly cell surface proteins, including those used for immune cell typing.

（2）Freely mixed TotalSeq ™ -A series antibodies, with the number of proteins depending on your selection;

（3）Freely mixed self-conjugated antibodies. For example, you can use the Stereo-seq self-conjugation kit to conjugate your primary antibodies of interest with antibody-derived-tag (ADT) oligonucleotides. After verifying that these antibodies are compatible with the Stereo-CITE workflow, you can freely mix them to achieve the desired number of proteins.