Always keep fluorophore-conjugated secondary antibodies in the dark exposure to too much light can lead to photobleaching.ĭeparaffinization may be insufficient (if tissue is embedded in paraffin).The fluorophore (if using fluorescent detection) may have been damaged by too much light exposure Use signal amplification to maximize the signal, eg a biotin-conjugated secondary antibody.The protein of interest is present in low abundance Check the scientific literature to see if protein is expected in the tissue type.The protein of interest isn't present in the tissue Check the storage instructions for your products on the datasheet.

Test the antibody in a native (non-denatured) western blot to make sure it is not damaged.Īntibodies or amplification kits may have lost activity due to improper storage and handling.Check the antibody datasheet to see if the antibody has been validated in the type of IHC you are using (eg formalin/PFA fixation, fresh frozen, etc).The antibody may not be suitable for IHC procedures which reveal the protein in its native state Incubate the sample for longer with the antibody (eg overnight) at 4☌.Add a higher concentration of primary antibody.Not enough antibody is bound to the protein

Make sure you use a secondary antibody that was raised against the primary antibody species.

Make sure that the isotypes of the primary and secondary are compatible.

Negative control for enzyme experiment