Frequently Asked Questions about TSA Multiplex Immunofluorescence Experiments
1. Can frozen floating sections be used for TSA experiments?
TSA kits are not suitable for floating sections. Because floating sections are too thick, which is not conducive to reagent penetration. Floating sections may undergo morphological changes during staining, such as curling and wrinkling.
2. How should antibodies be selected for mIHC on paraffin sections?
Try to use monoclonal antibodies. The antibody application should be IHC/mIHC/IHC-P. Priority should be given to antibodies that have been validated by knockout experiments.
3. Why does non-specific binding occur?
①If polyclonal antibodies are used, non-specific binding is likely to occur. This can be improved by switching to monoclonal antibodies, reducing the concentration, or decreasing the retrieval intensity.
②Signal amplification is too strong. Reduce the staining solution reaction time or concentration.
③Primary antibody concentration is too high or antigen retrieval is excessive. Use a lower antibody dilution ratio or reduce the antigen retrieval intensity.
4. How to avoid section detachment?
①Slides are not treated for adhesion prevention. Use adhesion-preventing slides or treat slides for adhesion prevention.
②Tissue fixation is incomplete or dehydration is insufficient. Re-optimize sample pretreatment methods, such as extending fixation and dehydration time.
③Excessive heat antigen retrieval treatment. Use microwave retrieval with gentle conditions, control the retrieval temperature at around 80°C or lower, and use 2× citrate buffer (pH 6.0) as the retrieval solution.
④Elution buffer temperature is too high. Reduce the elution buffer temperature or shorten the incubation time.
5. Why does color crosstalk occur?
①It may be related to the filter bandwidth of the imaging device. Try to use filters with narrow wavelength bandwidths.
②It may be related to incomplete elution of the previous round of antibodies. For antibodies with high affinity, such as CK, the antibody elution conditions need to be extended (increase elution temperature and time, etc.).
③It may be related to signal imbalance. For example, two adjacent channel dyes have one with too high intensity and one with too low intensity, causing the strong signal to overflow.
④Other possible reasons, such as mixed-up antibodies, forgotten elution/retrieval in the next round, etc.
6. For mouse samples, can mouse-origin primary antibodies be used?
It is generally not recommended. Because if the primary antibody is derived from mouse, the secondary antibody also needs to be anti-mouse, which may cross-react with endogenous IgG in the sample, leading to non-specific binding.
If it is necessary to use a primary antibody from the same species as the sample, it is recommended to perform a negative control experiment, that is, add only the secondary antibody without the primary antibody to detect whether non-specific signals will be generated.
If it is necessary to use a primary antibody from the same species as the sample, it is recommended to perform a negative control experiment, that is, add only the secondary antibody without the primary antibody to detect whether non-specific signals will be generated.
7. How to choose the order of markers/antibodies when doing multi-label staining?
Antibodies that are difficult to elute should be placed in the last round, otherwise color crosstalk is likely to occur. Alternate between rabbit and mouse primary antibodies. Markers that are more difficult to stain should be placed in the first few rounds. For low-expression targets, choose dyes with high fluorescence intensity and strong anti-quenching properties, and place them in the first few rounds. For high-expression targets, choose dyes with moderate fluorescence intensity to avoid signal saturation masking details. If two proteins are co-expressed, it is recommended to choose dyes with large wavelength differences.
8. Can traditional fluorescence staining be combined with TSA multiplex immunofluorescence?
Yes, but it is recommended to arrange traditional fluorescence staining in the last round. This is because during the antibody elution process in the TSA method, the primary and secondary antibody conjugates are eluted simultaneously.
9. What are the reasons for high background in bone tissue staining?
①Insufficient or excessive fixation. Insufficient fixation leads to antigen diffusion, while excessive fixation may cause non-specific binding. Solution: Optimize fixation time (usually within 24 hours with 4% paraformaldehyde), avoid using acidic decalcifying solutions that destroy antigens.
②Strong acid decalcifying solutions (such as hydrochloric acid, nitric acid) may destroy antigen epitopes or increase non-specific binding. Solution: Use EDTA decalcifying solution instead.
③High antibody concentration leads to high background. Solution: Reduce antibody concentration.
④Excessive dye development time. Solution: It is recommended to develop color for 5 minutes first, and the development time can be extended if the signal is weak.
10. Is light protection required during experimental operations? How long can stained slides be stored?
The fluorescent dyes in the Enklife TSA kit have excellent anti-quenching properties, so there is no need to operate in the dark under fluorescent lights, and there is no need to work in a dark environment during use. However, please avoid exposing the kit to direct sunlight. Stained slides can usually be stored at 4°C for 6 to 12 months.
11. Precautions for cell climbing slides in TSA multiplex immunofluorescence
①Cell climbing slides do not require antigen retrieval.
②Antibody elution can be performed gently with antibody elution buffer
③Antibody elution time should not be too long, otherwise the nucleus cannot be labeled
④Choose antibodies suitable for IF/ICC for cell climbing slides
12. Precautions for frozen sections in TSA multiplex immunofluorescence
①It is recommended to use paraffin sections as much as possible
②Fresh tissue adheres more firmly than fixed tissue
③Frozen sectioning has a relatively high risk, especially for sections thicker than 15μm
④Antibody elution can be performed gently with antibody elution buffer
⑤It is recommended to limit frozen sections to three labels or fewer
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