The single-color immunofluorescence image obtained from the fluorescence microscope shows the combined signals of multiple proteins, making it unsuitable for determining the spatial location of individual proteins.

The fluorescent imaging of secondary antibody staining is an important QC step in the biochemistry workflow. We strongly recommend that customers perform this step to ensure that primary antibodies successfully bind to the target antigens on tissues. If users choose not to take images, they can skip the secondary antibody incubation and imaging steps. However, STOmics will not provide after-sales service or compensation for any experimental result abnormalities caused by skipping this step.

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.

Repeatedfreeze-thaw cycles 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.

Stereo-CITE is compatible with three types of protein antibody panels for detecting different numbers of proteins.

(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 protein number of your choice;

(3) Freely mixed self-conjugated antibodies. For example, you can follow the Stereo-CITE self-conjugation protocol 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.

For Stereo-seq mIF experiments, Triton X-100, which may affect cell permeability, is added into blocking solution for antibody incubation before permeabilization; thus, the permeabilization time of should be specifically optimized.