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Effects of fixative and embedding medium on morphology and immunostaining of the cochlea
Jennifer T O'Malley1, Saumil N Merchant, Barbara J Burgess
1Massachusetts Eye and Ear Infirmary, Boston, Mass. 02114, USA. Jennifer_O'Malley@meei.harvard.edu
Insights
Celloidin provides superior morphology preservation for immunostaining in mouse cochlea studies. This embedding medium, combined with specific fixatives, optimizes antigen detection for otologic research.
Area of Science:
- Otolaryngology
- Immunohistochemistry
- Cell Biology
Background:
- Immunostaining is crucial for studying otologic disorders.
- Fixatives and embedding media can hinder antigen accessibility, impacting immunoassays.
- Optimizing tissue processing is vital for accurate protein localization.
Purpose of the Study:
- To systematically evaluate fixative and embedding medium effects on mouse cochlear morphology and immunostaining.
- To identify optimal processing conditions for reliable antigen detection in otologic research.
Main Methods:
- Tested three fixatives: formaldehyde (F), formaldehyde + acetic acid (FA), and formaldehyde + acetic acid + glutaraldehyde (FGA).
- Evaluated three embedding media: paraffin, polyester wax, and celloidin.
- Assessed morphology via light microscopy and immunostaining with six antibodies.
Main Results:
- Celloidin yielded superb morphology, polyester wax adequate, and paraffin suboptimal.
- Immunostaining was successful across all fixatives and embedding media.
- FA and FGA fixatives provided the most uniform immunostaining results.
- Celloidin and paraffin showed minimal differences in immunostaining quality for a given fixative.
Conclusions:
- Celloidin is the preferred embedding medium for optimizing both morphology and immunostaining in otologic studies.
- FA and FGA fixatives offer consistent results for antigen detection.
- Careful selection of fixatives and embedding media is critical for successful immunostaining in the cochlea.
Abstract:
The localization of proteins by immunostaining is a powerful method to investigate otologic disorders. However, the use of fixatives and embedding media (necessary for the preservation of morphology) can obscure antigens, making it difficult to perform immunoassays. We performed a systematic investigation of the effects of fixative and embedding medium on morphology and immunostaining of the mouse cochlea. Three different fixative solutions [4% formaldehyde (F), 4% formaldehyde + 1% acetic acid (FA), and 4% formaldehyde + 1% acetic acid + 0.1% glutaraldehyde (FGA)] and 3 different embedding media (paraffin, polyester wax, and celloidin) were used. Morphology was assessed using light microscopy. Immunostaining was studied using a panel of 6 antibodies (to prostaglandin D synthase, aquaporin 1, connective tissue growth factor, 200-kDa neurofilament, tubulin and Na(+),K(+)-ATPase). Preservation of morphology was suboptimal with paraffin, adequate with polyester wax and superb with celloidin. Immunostaining was successful using all 6 antibodies in all 3 fixatives and all 3 embedding media. While there were differences in strength of signal and localization of antigen between the 3 fixatives, overall, FA and FGA gave the most uniform results. For a given fixative and antibody, there was surprisingly little difference in the quality of immunostaining between celloidin and paraffin, while results in polyester wax were not as good in some cases. These results suggest that celloidin may be the embedding medium of choice for both morphological and pathological studies, including immunostaining when morphology must be optimized.
