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A positive control is needed to monitor the test validity. A negative control is needed to establish a baseline or "zero", to prove that the test is the reason for the measured effect.
NBT/BCIP substrate for AP is the most sensitive but, not widely used because the reaction is slow, does not allow adequate nuclear counterstain, the signal can diffuse and is not compatible with permanent mounting media. Development using DAB substrate for HRP is far more common due to speed of reaction, precise deposition and acceptable color contrast with nuclear stains.
IHC is relatively light-insensitive and allows the visualization of tissue architecture. Autofluorescence can sometimes make IF studies impossible. IF allows for staining of the same subcellular structure with different fluorophores.
Upon fixation, all intracellular trafficking of molecules is stopped. Drying and lipid solvent treatments (acetone, ethanol, etc.) create massive holes in the greater sub/cellular structure. This allows antibodies to cross membranes (extracellular, nuclear, etc.) in fixed cells. There is no detergent that causes an antigen that was previously hidden (masked) to be exposed (unmasked). Low levels of detergents such as Tween 20 in the washing solutions reduce the surface tension and allow the tissues/cells to remain wet.
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Incubation for too short will not produce adequate signal. Incubation for too long can result in negative (unspecific) staining. Since the final result of your technique can only be determined at the end of your technique when no corrections can be made, the dilution and time of incubation for each antibody should be determined individually. In general, antibodies with known high affinity should be used at high dilution and overnight incubations. Antibodies with various affinities (polyclonal) must be experimented with to determine optimum time and dilution.
Each freezing cycle causes aggregation of the immunoglobulin and loss of titer. Avoid freezing aliquots that you expect to use out in the next couple of years, unless they are small aliquots which you will use completely within 1 or 2 experiments. You may freeze reference reagents in small aliquots for reference or reagents to be stored without preservative.
Antibodies diluted as directed are very stable. Months and probably years. One study has sown extended shelf life for commercial reagents, beyond the manufacturer's expiration date. For antibodies used very diluted against low level antigens (<2K molecules/cell) is probably safer make fresh solutions monthly.
Reconstitute in an equivalent volume to the weight you purchased, so that the final concentration is 1 mg/ml. For example, if you purchased 100 µg of antibody, resuspend in 100 µl of pure distilled water. Dissolve the entire contents in water by mixing and lightly centrifuging at room temperature. It is possible to dissolve the contents in larger volume (200-500 µl) but the subsequent dilution may have to be adjusted. Insoluble lipoproteins may aggregate on storage. These do not interfere with the solubility of the antibody, spin down and use the clear supernatant.