One. no signal
1. Primary antibody does not match secondary antibody
Solution: In which animal species the primary antibody is produced, the secondary antibody should be selected against the corresponding species (for example, if the primary antibody is produced by rabbit, then the secondary antibody against rabbit should be selected).
2. Insufficient amount of primary antibody combined with the target protein
Solution: Increase the concentration of primary and secondary antibodies. Extend the incubation time at 4oC (eg overnight).
3. Antibodies are not suitable for tissue immunochemical assays where proteins maintain a natural 3-dimensional structure in tissue immunochemistry assays
Solution: The antibody was first identified by a reduced immunoglobulin (note: not a denatured Western blot).
4. The primary/secondary antibody/amplification kit may be inactivated due to improper storage, incorrect dilution or multiple freeze-thaw cycles.
Solution: Use a positive control to ensure that the primary/secondary antibody is normal.
5. The tissue of the study does not contain the target protein
Solution: Use a positive control recommended by the antibody supplier.
6. The amount of target protein expressed in the tissue being studied is small
Solution: Add one step to amplify the signal.
7. The secondary antibody is not protected from light.
Solution: Avoid secondary antibodies.
8. Dewaxing is not sufficient
Solution: Extend the dewaxing time and switch to new xylene.
9. The immobilization process may block the epitope
Solution: Expose the antigen with antigen retrieval techniques. Reduce the fixed time. Insufficient fixation results in strong staining at the edges of the sample, but there is no signal at the center of the sample.
10. The target protein is located in the nucleus, so that the antibody cannot bind to it.
Solution: Add a penetrant to the wash solution and / or antibody dilution. Tween 20 can be used as a penetrant, but the Triton X100 is stronger. The concentration of the penetrant is determined by the thickness of the slice.
11. PBS buffer is contaminated with bacteria that can destroy the phosphate groups on the target protein.
Solution: Add 0.01% azide to the PBS antibody preservation solution or use sterilized PBS.
12. Exposure time is too short
Solution: Extend the exposure time during signal acquisition.
two. Background is too high
1. not closed or closed completely
Solution: Increase incubation time and / or increase serum concentration in the blocking solution. Replace the blocking solution. We recommended blocking the sections with 10% normal serum for 1 hr and blocking the cultured cells with 1-5% BSA for 30 min.
2. The concentration of primary antibody may be too high
Solution: Reduce the concentration of primary antibody.
3. Incubation temperature is too high
Solution: Incubate sections or cells at 4oC.
4. Secondary antibody non-specific binding (antibody is destroyed)
Solution: Use a secondary control without primary antibody.
5. Fixative residue
Solution: Wash thoroughly with TBS or PBS between all steps.
6. Endogenous peroxidase is active
Solution: Use an enzyme inhibitor (such as inhibition of alkaline phosphatase with 2 mM levamisole or 0.3-3% by volume of hydrogen peroxide to inhibit peroxidase).
7. The immobilization process is too strong to change the antigen recognition site that the antibody can recognize.
Solution: Change the antigen retrieval technology and reduce the incubation time with the antigen unmasking solution.
8. Over-expansion
Solution: Reduce the incubation time during amplification and dilute the amplification kit.
9. (when using enzyme detection) used too much substrate
Solution: Reduce substrate incubation time.
10. (when using enzyme detection) chromogen reacts with PBS in tissues or cells
Solution: Wash the sections with TBS buffer before incubating with the substrate. The sections/cells were then washed with TBS buffer after incubation with the substrate.
11. Penetrant treatment destroys cell membrane and removes membrane protein
Solution: Do not add a penetrant to the buffer.
three. Non-specific staining
1. Primary antibody / secondary antibody concentration is too high
Solution: Try reducing the concentration of antibody and/or incubation time. Controlled with the signal intensity of cells that do not express the protein of interest.
2. Endogenous peroxidase is active
Solution: Use an enzyme inhibitor (such as inhibition of alkaline phosphatase with 2 mM levamisole or 0.3-3% by volume of hydrogen peroxide to inhibit peroxidase).
3. The stained tissue is derived from the species producing the primary antibody (eg, using mouse primary antibodies on mouse tissues). When a secondary antibody is used, since the secondary antibody is anti-mouse, the secondary antibody binds to all tissues.
Solution: Select antibodies produced from species that differ from the tissue source being studied.
4. Slice / cell dried
Solution: Store the sections/cells in high humidity without drying them.
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