Can Frozen float be used for TSA experiments?

The TSA mIHC kit is not suitable for floating chips

1. The thickness of the floating sheet is too thick, which is not conducive to the penetration of reagents;

2. The morphology of the float chips will change during the dyeing process, such as curl and crease.

The sample is a mouse. Can I choose the primary antibody from the mouse source?

This is usually not recommended because if the primary antibody is from a mouse, the secondary antibody will also need to be an anti-mouse antibody, which may cross-react with the IgG present in the sample, leading to non-specific binding.

If it is necessary to use primary antibody of the same species as the sample, it is recommended to carry out a negative control experiment, that is, without adding primary antibody, only add secondary antibody, to detect whether non-specific signals will be produced.

Can traditional fluorescence staining and TSA multiple immunofluorescence be used together?

Yes, but traditional fluorescence staining should be done in the last round. This is because the TSA method will elute both primary and secondary antibodies during antibody elution.

How to choose blocking buffer?

Commonly used blocking buffer include bovine serum and goat serum. It is recommended to choose serum that matches the host species of the secondary antibody.

The antibody diluention buffer provided in our plus mIHC kit contains a variety of protectants and preservatives, which can also be used for blocking.

Is there a problem if the antibody eluent is cloudy?

When the ambient temperature is low, crystals may precipitate in the antibody elution solution. To solve this problem, the elution solution can be heated in a constant temperature box or water bath at 37℃ before use, and then it can be used after it returns to its transparent state.

Can TSA be performed on cell slides, frozen sections or frozen drifts?

TSA experiments can be performed on cell slides and frozen sections, but it is recommended to limit the number of markers to three or less. There are dedicated kits available for use, please contact us, but more markers are not recommended.

matters need attention:

1. Frozen sections made from fresh tissue are more firm and less likely to fall off than fixed tissue.

2. Wax sections are recommended as a priority because frozen sections are usually thicker and prone to dissection during multiple rounds of antibody elution (especially when the thickness exceeds 15μm), which can affect the observation of indicators.

Do kit operations need to be lightproof?

The fluorescent dye in our kit has excellent anti-quenching properties, so there is no need to avoid light under fluorescent lamps, and there is no need to use in dark environment. However, please be careful not to expose the kit to direct sunlight.

How long the fluorescent signal will last?

The stained slides in our laboratory can usually be stored for 6 to 12 months under 4℃ conditions.

What is the significance of the order of antibody selection in mIHC experiments?

Based on Neorise laboratory experience, the following strategies are recommended:

1. Start the preliminary experiment with those that have poor priority specificity and good secondary specificity.

2. According to the results of the preliminary experiment, the order needs to be adjusted

The hard-to-get rid of antibodies are at the end of the list;

3. Put the more difficult indicators in the first few rounds (such as FOXP3)

4. Rabbit mouse primary antibody cross-reactive

5. The repair/elution method needs to be adjusted according to the primary antibody

The first round is usually used for indicators suitable for EDTA 9.0 repair; according to our experience, the first round is usually used for EDTA 9.0/ high pressure 6.0, the second round and later are usually used for 6.0, and multiple resistance is recommended for 6.0.

Non-specific production and improvement methods?

1. Antibodies are polyclonal and easy to produce non-specificity, which can be improved by switching to monoclonal antibodies or reducing concentration and repair intensity;

2. The signal amplification is too strong, which can reduce the reaction time or concentration of dye;

3. If the concentration of primary antibody is too high or the repair is excessive, use a relatively low dilution ratio of antibody, or reduce the repair intensity (temperature or time or PH of repair solution)

Why do colors strung together?

Color matching is related to many factors:

1. It may be related to the bandwidth of the filter of the imaging device, so try to choose a narrow wavelength band filter;
2. It may be related to the incomplete elution of the previous round of antibodies. For some antibodies with high affinity, such as CK, the antibody elution conditions need to be extended (increase the elution temperature time, etc.);
3. It may be related to signal imbalance, such as adjacent channel dyes, one is too high, the other is too low, resulting in strong signal overflow;
4. Other possible causes, such as antibody confusion, the next round of antibodies forgetting to elute/repair, etc.