7. No positive immunostaining obtained. Antigen does not survive processing—check antigen's sensitivity to fixation and dehydration using fresh frozen, and fixed, cryoprotected, frozen sections. Antigen is not present in tissue—use a positive control. Primary antibody is at fault—use a positive control and a new aliquot or batch. Secondary antibody or detection system is at fault—check potency of secondary antibody using an alternative primary antibody. If using an indirect method of detection, this may not be sensitive enough—try an amplification system.
8. Patchy immunostaining obtained. Gelatin solution crept over surface of section during decompression—remount new sections with additional care. Nonuniform fixation—reassess fixation protocol. Sections allowed to dry out during staining—restain using an adequate volume of immunoreagent and ensure humidity of chamber.
9. Background staining too high. Concentration of primary antibody too high—reduce titer to minimum. Secondary antibody is interacting with the tissue-e.g., for mouse monoclonals used on rat tissue, use rat adsorbed antimouse IgG, or use control lacking primary antibody. Tissue has (a) autofluorescence or (b) endogenous enzyme, e.g., peroxidase—(a) View section without antibodies or (b) react section with enzyme substrate alone. Antibodies binding via ionic interaction—increase pH from 7.4-8.2., increase salt concentration, use detergent, e.g., 0.1% Tween-20.
10. Immunofluorescent stain is rapidly lost from the section during microscopy. Antibody (probably primary) is labile in mountant—refix sections in 4% PF for 5 min after immunostaining and prior to mounting in glycerol-based mountant.
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