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105results:
Q Do Biolegend antibodies with a Poly A tag compete against target mRNA molecules on Stereo-seq chip? As the target protein number increases, does this enhance the competition for capture probes between the PolyA on antibodies and the mRNA from the tissue?
A

In theory, competition exists. However, there are sufficient probes on each DNB spot to effectively mitigate this competition. It has been confirmed that there is no impact on mRNA capture in a comparison of two adjacent tissue sections: one section with no antibodies applied, and the other with a high-plex antibody panel (for example, the 100+ plex Biolegend antibody panel) applied.


Q How to distinguish the background signals from the signal in a protein expression heatmap?
A
It should be determined based on existing knowledge of each protein expression pattern in cells within the relevant field.
Q How can we determine the spatial location information of individual proteins in a single-color immunofluorescence image containing multiple proteins?
A

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.


Q Concerning the secondary antibody incubation and imaging process, can fluorescent imaging of secondary antibody staining be omitted without impacting the experimental results? If imaging can be omitted, is it also possible to skip the secondary antibody incubation?
A

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.

Q When imaging the secondary antibody during the Stereo-CITE Proteo-Transcriptomics workflow, is the fluorescent imaging based on multiplex immunofluorescence or single-color immunofluorescence? What is its purpose?
A

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.


Q What is the purpose of using sheared salmon sperm DNA?
A

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.


Q Can 4% PFA be repeatedly frozen and thawed for use?
A

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.


Q The current protocol uses PFA to fix the sections at room temperature after tissue sectioning. Can the sample be fixed with PFA before OCT embedding for subsequent experiments?
A
Currently, the Stereo-CITE Proteo-Transcriptome protocol is not compatible with PFA-fixed frozen samples and is only suitable for fresh frozen samples.


Q What types of antibody panels are Stereo-CITE product compatible with?
A

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.

Q Does omitting the primary and secondary antibodies, along with the Fc blocking reagent, during the permeabilization optimization affect the determination of the optimal permeabilization time?
A
No, it won't affect the results.
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