1, 染好的片子保存时间？

我司实验室的染色玻片在4℃条件下通常可保存6至12个月。

2,能否使用冰冻漂片进行TSA实验？

TSA试剂盒不适用于漂片

1.漂片厚度太厚，不利于试剂的渗透；

2.漂片在染色过程中形态会发生改变，如卷曲，褶皱等

3，样本为小鼠，是否可以选择小鼠来源的一抗？

通常不建议采取这种方式，因为如果一抗来源于小鼠，那么二抗也需使用抗小鼠的二抗，这可能会与样本中本身存在的IgG发生交叉反应，从而导致非特异性结合。

若必须使用与样本同种属来源的一抗，建议进行阴性对照实验，即在未添加一抗的情况下，仅加入二抗，以检测是否会产生非特异性信号。

4，传统荧光染色和TSA多重免疫荧光能否搭配使用？

可以，但建议将传统荧光染色安排在最后一轮进行。这是由于TSA法在抗体洗脱过程中，会同时洗脱一抗和二抗的结合物。

5，如何选择封闭液？

常用的封闭液包括牛血清和山羊血清，建议选择与二抗宿主种属相匹配的血清。

我司的plus版试剂盒中提供的抗体稀释液含有多种保护剂和防腐剂，也可以用于封闭。

6，试剂盒操作需要避光吗？

我司试剂盒的荧光染料具有优异的抗淬灭性能，因此无需在日光灯下进行避光操作，使用过程中也无需在暗环境中进行。不过，请注意避免试剂盒暴露在太阳光下直射。

7，多标实验中抗体的选择顺序有何讲究？

根据我们实验室的经验，推荐以下策略：

1、优先特异性差的、其次特异性好的开始预实验。

2、根据预实验结果，需要调整顺序，难以洗脱的抗体，摆在最后一轮；

3、比较难做的指标放在前面几轮做（比如 foxp3）

4、兔鼠一抗交叉做    

5、修复/洗脱方式是需要根据一抗调整条件的

第一轮通常做适合 EDTA 9.0 修复的指标；根据我司经验，第一轮通常采用 EDTA 9.0/高压 6.0  第二轮及以后通常采用 6.0 ，多抗建议 6.0  。

8，非特异性产生与改善方法？

1.抗体是多克隆的，容易产生非特异，可以换用单克隆抗体或者降低浓度以及修复强度来改善；

2.信号放大过强，可以降低染液反应时间或者浓度；

3.一抗浓度过高或者修复过度，采用比较低的稀释抗体比例，或降低修复强度（温度或者时间或者修复液 PH）

9，为什么会串色？

串色与多种因素有关系：

1. 可能与成像设备滤光片带宽有关系，尽量选用窄波长带宽的滤光片；

2. 可能与上一轮抗体未被完全洗脱关系，对于一些亲和力比较高的抗体比如CK等，抗体洗脱条件需要延长（提高洗脱温度时间等）；

3. 可能与信号不平衡有关系，比如相邻两个通道染料，一个强度过高，一个过低，导致强的信号发生外溢；

4. 其他可能存在的原因，比如抗体弄混，下一轮抗体忘记洗脱/修复等。

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.