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Endogenous enzymes cause structural and chemical artifacts in methacrylate- and celloidin-embedded sections of
1Department of Fisheries and Allied Aquacultures, Alabama Agricultural Experiment Station, Auburn University 36849.
Abstract:
Bovine exocrine pancreas and fish (Rivulus ocellatus marmoratus) liver containing pancreatic acini were cryofixed, freeze-dried, and embedded in methacrylate or double-embedded in celloidin and paraffin. In chemically unfixed sections incubated in aqueous solutions, dissolution of zymogen granules was coincident with loss of tissue structure and antigenicity. Type II-S soybean protease inhibitor at 150 mg/liter during section flotation and in aqueous reagents used for immunohistochemistry prevented these artifacts and allowed the use of more dilute antibody solutions. Loss of glycogen from fish hepatocytes was most rapid in areas adjacent to pancreatic acini. Rapid loss of glycogen was attributed to amylase and was prevented by using poly-L-lysine instead of 3-aminopropyltriethoxysilane slide adhesive and by using alcoholic solutions during PAS staining. Inhibition of endogenous enzymes is an important consideration in the development of histological protocols with freeze-dried tissue sections.
Insights
Freeze-dried tissue sections require careful handling to preserve structure and antigenicity. Inhibiting endogenous enzymes during histological protocols is crucial for accurate results in immunohistochemistry and glycogen staining.
Area of Science:
- Histology
- Biochemistry
- Immunohistochemistry
Background:
- Cryofixation and freeze-drying are advanced techniques for preserving tissue ultrastructure.
- Chemically unfixed tissue sections are susceptible to artifacts during aqueous incubations, including antigen and structural loss.
Purpose of the Study:
- To investigate and mitigate artifacts in cryofixed, freeze-dried histological sections.
- To optimize protocols for immunohistochemistry and glycogen staining in sensitive tissues.
Main Methods:
- Bovine pancreas and fish liver tissues were cryofixed, freeze-dried, and embedded.
- Sections were incubated in aqueous solutions with and without protease inhibitors and alternative slide adhesives.
- Periodic acid–Schiff (PAS) staining was performed using aqueous and alcoholic solutions.
Main Results:
- Dissolution of zymogen granules and loss of antigenicity occurred in unfixed sections.
- Type II-S soybean protease inhibitor prevented zymogen granule dissolution and antigen loss.
- Glycogen loss from fish hepatocytes was prevented by using poly-L-lysine and alcoholic PAS staining solutions.
Conclusions:
- Endogenous enzyme activity is a significant factor causing artifacts in freeze-dried tissue sections.
- Protease inhibitors and specific staining solutions are essential for preserving tissue integrity and antigenicity.
- Optimized histological protocols are critical for reliable analysis of freeze-dried biological samples.