1. What are the Causes of Negative Immunohistochemistry Staining Results?
a. Antibody concentration and quality problems and wrong source selection of antibodies. It is not that the higher the antibody concentration, the more likely to be a positive result. The antigen-antibody reaction has prozone and posterior zone effects, and the optimal concentration must be explored.
b. Antigen restoration is incomplete. For formaldehyde-fixed tissues, sufficient antigen restoration must be used to open the antigen epitopes to facilitate binding to antibodies. Microwave repair is recommended, and high-pressure repair is also recommended if this is not possible.
c. The tissue section itself has a low content of this antigen.
d. Serum blocking time is too long.
e. DAB incubation time is too short.
f. Cell permeation is not complete, and the antibody cannot fully enter the cell to participate in the reaction.
g. To start immunohistochemistry, it is recommended to take a positive photo first to rule out problems such as antibodies.
2. Paraffin Section or Frozen Section?
a. Those who require frozen sections may not be able to do paraffin sections. Because paraffin sections need to be baked at high temperature, the antigenicity of the tissue may be destroyed. If the antigenicity of the tissue is relatively stable, paraffin sections can be used; however, if paraffin sections are required, frozen sections can be used.
b. The advantage of frozen section is that it can better preserve the antigen immune activity of the tissue, and the step of antigen retrieval is not required for immunohistochemistry. The disadvantage is that ice crystals are easy to form in the cells and destroy the cell structure, which may disperse the antigen. The slice thickness is thicker than that of paraffin, and the slices made are not as beautiful as paraffin. When you buy a primary antibody, it is written in the catalog what kind of sections to make. If it says that it can only be frozen, it cannot be used for paraffin. If it says that it can be used for both, then it can be done.
c. The advantage of the paraffin section is that it can maintain the morphology and structure of tissue cells, and it is easy to store at room temperature. However, frozen sections are more troublesome and must be stored in a low-temperature refrigerator at -80°C, especially for in situ hybridization. Consistent preservation is important to prevent RNA degradation. Since the paraffin section can be cut to about 4 microns, the in situ hybridization probe is easy to penetrate into the tissue, and it is easy to succeed, and the color/morphology obtained is better than that of the frozen section.
3. How to Choose a Primary Antibody?
a. Selection of monoclonal and polyclonal antibodies: Specific antibodies produced by one clone are called monoclonal antibodies. Monoclonal antibodies can specifically bind to a single specific antigenic determinant, like a missile hitting a target with precision. On the other hand, even for the same antigenic determinant, antibodies can be produced by several clones in the body, forming a mixture of several monoclonal antibodies, called polyclonal antibodies. In the antigen-antibody reaction, monoclonal antibodies generally have strong specificity, but relatively low affinity, and relatively low sensitivity for detecting antigens; while polyclonal antibodies are slightly weaker in specificity, but have a strong affinity and high sensitivity, but are prone to non-specific staining (can be avoided with closures, etc.).
b. Choice of application range: Some primary antibodies can only be used for Western blotting, or immunohistochemistry, immunofluorescence, immunoprecipitation, etc., even marked paraffin sections or frozen sections.
c. Species reactivity: This is very important, indicating that there may be species differences in this antibody, and this antibody is suitable for detecting antigens in which species of animals.
d. Species source: Generally, polyclonal antibodies are mostly derived from rabbits, and monoclonal antibodies are mostly derived from mice, but there are exceptions. Select the corresponding secondary antibody according to this source.
e. The choice of manufacturer.
4. What is the Selection Principle of Blocking Serum?
a. The blocking serum is generally from the same source as the secondary antibody. The animal's own antibody in the serum can bind to the cross-reactive site in the tissue in advance. Otherwise, if it binds to the secondary antibody in the subsequent steps, it will cause background.
b. Calf serum, BSA, goat serum, etc. can also be used, but the source of the primary antibody should not be the same.
5. How to Analyze the Results of Immunohistochemistry?
a. Positive staining cell counting method: Under a 40× light microscope, randomly select 10 non-overlapping fields of view, count positively colored cells manually or by machine, and take 3-6 slices of different animal tissues in each group, and then compare between groups.
b. Gray density analysis: By selecting the same area on different groups and different animal tissue sections, using Image J for gray density analysis under the same conditions, and then performing statistical analysis.
c. Scoring method: Score the tissue sections under the light microscope according to the degree of staining (0-3 points are negative staining, light yellow, light brown, dark brown) and the positive range (1-4 points are 0-25%, 26-50%, 51-75%, 76-100%), you can finally add up the scores and compare them.
The above methods have their own advantages and disadvantages, please choose carefully. To get the right result, you need to make high-quality slices with uniform coloring and light background.
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