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Symptom
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Issue
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Recommendations
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No signal
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Antibody Application
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Increase the concentration or incubation time of the primary or secondary antibody.
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Tissue Fixation
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Over fixation can cause epitope masking. Decrease the time or concentration of the fixative.
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Antibody compatibility
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Confirm that your primary and secondary antibodies are compatible by checking the species reactivity.
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Confirm the antibody can be used for assays in which the protein is in its native conformation.
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Ensure that the secondary is working and compatible with your primary.
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Permeabilization
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Use 0.5-1.0% Triton detergent in the buffers in order to allow full permeabilization of antibody and buffers into the tissue sections.
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Microscope Adjustments (Fluorescence)
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Increase the exposure time of your camera.
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Background
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Antibody Concentration
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Decrease the concentration of the primary/secondary antibody.
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Blocking
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Increase the incubation time or concentration of serum in the blocking buffer.
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Antibody Application
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Always incubate primary antibodies overnight at 4° C. Room temperature incubation increases unspecific binding and causes higher background.
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Confirm that the secondary is not crossreacting with the cells by performing the assay without the primary.
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DAB Reaction
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Do not overexpose the DAB reaction. Rinse DAB off slides sooner.
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Endogenous peroxidases are activating the reaction. Quench with hydrogen peroxide.
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ABC Method
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Endogenous biotin is activating the complex. Block with avidin/biotin blocking kit.
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Cells Drying
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Fluorescent signal will be lost if the cells are allowed to dry. Ring coverslips with nail polish.
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Microscope Adjustments (Fluorescence)
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Increase the exposure time of your camera.
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Spectral Overlap (Fluorescence)
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If double or triple labeling the cells, confirm that the secondaries do not overlap into the same spectral range
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Washing
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Increase the amount of washes. Add very gentle agitation to the plates.
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Sectioning Tissue
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Holes in the tissue
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Ensure the blade is adequately sharp. Adjust the cutting speed. Perfuse at a lower rate.
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Tissue Curling
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Decrease the temperature of the cryostat or open cryostat lid more. Cut thicker sections.
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Tissue Falling Off Slides
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Use coated slides. Place slides in covered dish with a small amount of 16% formaldehyde on the bottom to fix the tissues to the slides.
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