Immunostaining Fixative Requirements
For immunostaining (immunohistochemistry/immunoperoxidase), formalin-fixed tissue is the standard and preferred fixative, while for immunofluorescence studies, fresh-frozen tissue or Michel's solution is required. 1
Immunohistochemistry (IHC/IP) - Formalin is Standard
Formalin-fixed, paraffin-embedded tissue is the gold standard for immunohistochemistry and allows the full range of immunostaining to be performed. 1, 2
Neutral buffered 10% formalin provides excellent preservation of morphological detail, produces tissue cellular appearance well-recognized by pathologists, remains stable at room temperature, and critically allows subsequent immunohistochemistry and molecular studies. 2
The material for immunoperoxidase (IP) staining is taken directly from the formalin-fixed material also used for light microscopy, requiring no special tissue preparation. 1
Modern antigen retrieval techniques have made formalin-fixed tissue highly suitable for immunostaining, with well-developed procedures that provide reproducible results and overcome the antigen masking that occurs during formalin fixation. 1, 3, 4
Immunofluorescence (IF) - Requires Fresh or Michel's Solution
For immunofluorescence studies specifically, the tissue must NOT be exposed to formalin and should either be fresh-frozen or placed in Michel's fixative. 1, 2, 5
The portion designated for IF must be picked up with a clean tool and placed in transport solution (tissue culture medium or normal saline), remaining completely untouched by formalin or other fixatives. 1
Michel's medium (Modified Michel's Tissue Fixative) is the preferred transport medium when specimen delivery to the laboratory may be delayed beyond 24 hours, as it preserves tissue for immunofluorescence studies during extended transport times. 1, 5
Normal saline (0.9% NaCl) is optimal for DIF specimens when transport time is under 24 hours, providing superior results by reducing background fluorescence and enhancing specific immunoreactant staining. 5
Critical Distinction Between Techniques
The key difference is that immunohistochemistry/immunoperoxidase (IP) uses formalin-fixed tissue, while immunofluorescence (IF) requires unfixed or Michel's-fixed tissue. 1
If the pathology laboratory uses IP instead of IF, biopsy handling is greatly simplified because the material for IP is taken from the formalin-fixed material also used for light microscopy. 1
IP has almost completely replaced IF in routine surgical pathology and many renal pathology laboratories because it allows correlation of light microscopy and immunostaining from a single formalin-fixed block. 1
Common Pitfall to Avoid
Never place tissue intended for immunofluorescence in formalin - this will destroy the ability to perform IF studies. 1 The specimen must be divided at the time of biopsy, with separate portions allocated for formalin fixation (for light microscopy and immunohistochemistry) versus fresh/Michel's fixation (for immunofluorescence). 1