Paraformaldehyde fixation is indeed a long-standing talk. When many graduate students are doing experiments, many brothers and sisters may ask them to fix it with paraformaldehyde. Some may be to preserve samples for a long time and hope to concentrate them. Dyeing, some people may not be able to queue up after dyeing, and want to maintain stable quality for a long time. So it was passed down by word of mouth.
Pre-fixation issues
When doing scientific research or clinical practice, many FLOWERs may habitually pre-fix specimens with 4% paraformaldehyde, possibly for three purposes: (1) For Inactivate microorganisms (bacteria, viruses, etc.) that may be present in the specimen to ensure safety. (2) It is hoped that the specimens can be stored for a long time. (3) Make preparations before intracellular staining. However, many people may have overlooked one point, that is, if the specimen is fixed with paraformaldehyde before staining, the conformation of some sites on the cell surface will change after fixation, making the antibody unable to bind and producing false negatives. It may also cause other sites to be damaged due to the fixation effect. spots produce nonspecific binding (false positives).
Therefore, it is very important to know which antibodies cannot be prefixed before staining. Fortunately, we don’t need to do the verification ourselves. On the Biolegend website [1], the considerate technical staff have done a series of tests for us. The following two tables, one is the human antigen and the other is the mouse antigen, can be viewed It turns out that most of the antibodies that cannot be pre-fixed are chemokine receptors. Therefore, everyone must be careful not to fix them first and then stain them during the experiment.
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Table 1. Effect of 4% paraformaldehyde prefixation on surface staining of various antibodies (human). The ticked ones indicate prefixation is allowed, and the crossed ones indicate prefixation. The fixation has a great impact
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Table 2. Effects of 4% paraformaldehyde prefixation on surface staining of various antibodies (mouse). The ones checked indicate prefixation is allowed. The crossed ones indicate that pre-fixation has a great influence
Post-staining fixation issues
The fixation after staining has little to do with the antigen conformation and more to do with fluorescein. In the past, when there were less than 4 colors, the fluorophores used were non-tandem, so most of them had little effect. However, with the emergence of various new tandem dyes, paraformaldehyde fixation has become a problem.
In fact, when you search for antibodies with tandem staining markers (such as PE-cy7, APC-cy7, etc.), you will find some more detailed descriptions that indicate (search the BD website for APC- Cy7 antibodies are used as examples):
Fixed cells should be analyzed within 4 hours of fixation in paraformaldehyde or transferred to a paraformaldehyde-free buffer for overnight storage.
This means using more Samples fixed with paraformaldehyde must be tested within 4 hours, otherwise the tandem dye may be easily degraded. If stored for a long time, it still needs to be washed and transferred to a buffer that does not contain paraformaldehyde.
It should be noted that students who do GFP fluorescence detection also need to pay attention to the fact that paraformaldehyde fixation will also affect GFP. Paul A Lucas of New York Medical College believes that this is due to the paraformaldehyde fixation. It does not quench the GFP fluorescence, but changes the conformation of the GFP protein, making it unable to produce fluorescence [2]. Sometimes the fluorescence you detect after fixation may be autofluorescence, or may be emitted by the remaining GFP that has not changed its conformation.
Solution
1. If you are testing molecules that are easily affected by paraformaldehyde in the above table, please try not to pre-fix it. If you need to preserve the sample for a long time, you can consider freezing the sample. nucleated cells.
2. If you are detecting molecules that are not affected by paraformaldehyde in the above table, you can pre-fix and preserve them with 1% paraformaldehyde, but they cannot be used for a long time, otherwise it will affect the cell FSC, SSC and fluorescence. Strength has a big impact.
At this time, you can consider using FlowGuard? Cell Preservation Solution [3] from FlowChinese.com. FlowGuard? is a domestic patented original product. It does not contain paraformaldehyde and can slowly release a special weakly fixed component. The changes in FSC are relatively small. The paraformaldehyde is mild and maintains the antigen strength for a longer period of time. Sample: preservation solution = 6:1 ~ 10:1. The test results are stable within 21 days.
3. If you want to keep the fluorescence stable for about 24 hours after dyeing, the first choice is to avoid light and 4 degrees. We have used 10 colors (BV405, KO, FITC, PE, ECD, PE- cy5.5, PE-cy7, APC, APC-A700, APC-cy7). During informal testing of the flow cytometry scheme, several cases have been measured. After being stored in the dark at 4 degrees for 24 hours, the fluorescence intensity has almost no change, and the cells Good grouping. If you are still worried, you can consider purchasing a commercial special preservation agent suitable for tandem dyes