1. No signal
Antibody causes
1. The problems of primary and secondary antibodies, poor specificity of primary antibody, or low concentration of primary and secondary antibodies, insufficient incubation time, etc. lead to weak binding. 

2. The primary antibody and the secondary antibody cannot recognize each other. If the primary antibody selects murine antibody, the secondary antibody selects anti-rabbit antibody or recognizes antibody of other species, the secondary antibody and the primary antibody cannot bind to each other. It is suggested that the experimental operation should be carried out in strict accordance with the instructions, and the pre-experiment can be carried out appropriately to explore the incubation concentration and time. 
Sample and Reagent Problems
3. Slice too stale
Different target proteins may have different degradation or even disappear for the binding activity of antibodies after being stored for a period of time. If the slices need to be stored, it is recommended to store them in a constant temperature environment of 4 ℃. 

4. Insufficient sensitivity of detection reagents, too short incubation time, etc. 

5. The cell climbing slice or tissue sections itself has low antigen expression or no expression. 
Experimental process issues
6. incomplete antigen retrieval
For paraffin sections for antigen repair is the key link of the experiment, antigen repair can use hot water bath, microwave oven or pressure cooker, protease repair, to ensure the best conditions of the buffer system, generally can be based on historical data and literature to choose, can also be carried out comparative experiments, groping repair experimental conditions. (For cell climbing slice, antigen repair is generally used 100 mm tris, 5% [w/v] urea, pH 9.5 for hot water bath for 10min, this step is optional to increase the combination strength of the antibody)

7. Fluorescence damage
Use the secondary antibody to avoid light, and select the appropriate fixation method when fixing the sample: according to the nature of the substance to be detected and the requirements of the immunofluorescence reagent, select the appropriate fixation method, such as ethanol fixation, glacial acetic acid fixation, etc. Selection of Appropriate Enzymatic Conditions: If necessary, the sample is treated using appropriate enzymatic conditions to maintain the integrity of the fluorescence. 

8. Fixed time is too long
Fixation is to prevent the diffusion of isolated tissue autolysis antigen, but improper selection of fixation reagents and inappropriate use of concentration, such as the use of paraformaldehyde, formaldehyde, fixation time is too long will lead to antigen damage, resulting in poor antibody binding. 

9. Insufficient cell permeability, resulting in insufficient antibody staining
Triton is commonly used in order to allow sufficient penetration of reagents and antibodies throughout the sample. Ensure that the slide is completely permeabilized, and the recommended concentration is 0.5 - 1%. If it is membrane surface antigen can be omitted. 

10. Wrong excitation wavelength
Ensure that the excitation wavelength matches the fluorophore. 

2. High background 
1. Sample high background
Blank controls or secondary incubation controls need to be set to prevent background, high background or non-specific coloration caused by autofluorescence of the sample. If the sample background can not be resolved to consider the use of fluorescence quencher: add fluorescence quencher, such as cesium bromide, acid leucine, etc., to reduce the interference of background fluorescence. 

2. Insufficient washing in the experimental process
Cell slides and tissue sections should be washed thoroughly after incubation with primary antibody or at the end of incubation with secondary antibody to prevent non-specific staining of primary antibody or secondary antibody.

3. Insufficient slice closure
Ensure that the Cell slides or tissue sections are completely sealed. Before the primary antibody is incubated, use a suitable sealing solution for sealing. The sealing solution is generally selected from the same species as the secondary antibody. If the secondary antibody is sheep anti-rabbit, the sealing solution should be sheep serum. 

4. Insufficient washing
Consider increasing the number of immersion times and extending the immersion time after antibody incubation to avoid excessive background coloration due to insufficient cleaning times or time. 

5. Background coloring problems caused by mechanical damage or incomplete dewaxing
During the dewaxing treatment, the temperature of the slide should be ensured to be appropriate, the solvent should be quickly put in, and the dewaxing antigen should be repaired to avoid the dry film phenomenon of the slide. The antibody should be incubated in a wet box to avoid the sample drying. 

6. Antibody concentration used too high
The use of antibody concentration is too high will cause the background value to deepen and non-specific staining. It is recommended to operate in strict accordance with the recommended concentration in the instructions. Concentration titration before the experiment can effectively prevent the improper use of concentration. 
3. Tissue section and exfoliation

If the tissue is detached during the staining process, it is mainly caused by the experimental process and the materials used, and the following conditions can be optimized:

1. Slides containing polylysine were used for tissue sections, and slides with polarity having high binding activity were used to increase the stability of section binding. (The Cell Climbing Tablets can choose to use polynonine or high adsorption capacity climbing tablets to prevent cells from falling off).

2. Appropriately increase the baking time and adjust the appropriate temperature. If large-scale stripping occurs, problems often occur in the experimental process, which can prolong the baking time and increase the baking temperature.

3. In addition to the above precautions, when washing with PBS during operation, it should be gentle as far as possible to avoid washing directly against the sample. It is recommended to choose leaching to reduce the film detachment caused by physical impact. 

4. When antigen retrieval is performed, a short thermal quench of temperature causes cell deflaking. 

5. The use of antigen thermal repair and microwave repair, the buffer burst caused by the release of the film. 

4. Cell /tissue morphology damage
Optimization of fixation conditions: Select the appropriate fixation method and fixation time to maximize the integrity of the cell or tissue structure. 
1. Adjust the staining temperature: Decreasing the staining temperature can reduce the damage of cell or tissue structure. 

2. Optimizing Antibody and Stain Selection: Select antibodies and stains appropriate for specific cell or tissue types to improve staining and minimize structural damage. 
5. Non-specific staining in Cell climbing slice or tissue slices
Antibody causes
1. Antibody use concentration is too high, antibody incubation time is too long easy to increase the background coloring. Non-specific binding can be effectively reduced by shortening the primary /secondary antibody incubation time and diluting the antibody concentration. 

2. Using monoclonal antibodies
Although polyclonal antibodies have the advantages of rich epitopes and strong affinity, there will also be non-specific binding and other phenomena. The use of monoclonal antibodies can effectively avoid non-specific collection caused by this reason. 

3. For some samples where the source species of the secondary antibody is the same as the source species of the test sample, it may contain FcR or the secondary antibody may bind endogenous IgG, resulting in a higher background. 
Sample issues
1. The sample itself has autofluorescence, and it is recommended to select an appropriate fluorescence channel or use a quencher for fluorescence quenching. 
2. There are other non-specific binding reasons for the sample, such as complement, charge, etc., which require increased blocking time and increased washing times. 

Reasons for the experimental process
1. Insufficient closure
Before the primary antibody is incubated, a suitable blocking solution is used for blocking. Generally, the blocking solution is selected from the same species as the secondary antibody. If the secondary antibody is sheep anti-rabbit, the blocking solution should be sheep serum. 

2. Sample dry sheet
In the whole experimental process, the incubation conditions should be strictly controlled to avoid dry chips caused by reagent evaporation and process. 

3. full washing
Increase the number of washes or use a more stringent wash buffer to reduce the occurrence of non-specific binding and false positive results. 

4. Acquisition image parameters
Choosing appropriate excitation intensity, exposure time, etc. has a certain optimization effect on false positive and non-specific binding.