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Producing frozen sections of calcified bone
H H McElroy1, M S Shih, A M Parfitt
1Bone and Mineral Research Laboratory, Henry Ford Hospital, Detroit, Michigan 48202.
Abstract:
We describe a procedure for the rapid production and maintenance of fresh frozen bone biopsies which can be used for a variety of immunohistochemical techniques. Within 5 min of excision, tissue is placed in cold 5% polyvinyl alcohol, surrounded with 3% carboxymethylcellulose in a hand made aluminum foil embedding mold and frozen by immersion in an absolute ethanol/dry ice slurry at -70 C. The tissue block is attached to the specimen stub with cryocompound and installed in a -32 C cryostat whose tungsten carbide D profile knife is maintained at -70 C. Automatic controls are set at a slow cutting speed and the "sectioning window" is adjusted to fit the biopsy size. Knife angle, thickness gauge and antiroll bar are changed to produce a complete section. The block face is smoothly "papered" with a polyvinylpyrrolidone (PVP) impregnated Ross lens paper strip. A single section is cut and positioned on a sequentially numbered, acid cleaned, double dipped chrome-alum gelatin coated slide; adhesion is aided by "press-blotting" with bibulous paper. Sections are stored at -20 C or in a desiccator at room temperature. A brief fixation followed by removal of the water soluble PVP and lens paper generates fresh frozen bone sections suitable for further analysis.
Insights
This study presents a rapid method for preparing fresh frozen bone biopsies for immunohistochemistry. The technique ensures high-quality tissue sections suitable for various diagnostic and research applications.
Area of Science:
- Histopathology
- Biomedical Engineering
- Tissue Processing
Background:
- Fresh frozen tissue sections are crucial for accurate immunohistochemical analysis.
- Existing methods for bone biopsy preparation can be time-consuming and may compromise tissue integrity.
Purpose of the Study:
- To develop and describe a rapid, reproducible procedure for preparing fresh frozen bone biopsies.
- To ensure the suitability of prepared sections for diverse immunohistochemical techniques.
Main Methods:
- Bone tissue is rapidly embedded in polyvinyl alcohol and carboxymethylcellulose, then frozen at -70°C.
- Cryostat sectioning is performed at -32°C with specialized knife and antiroll bar adjustments.
- Sections are adhered to slides using a polyvinylpyrrolidone (PVP)-impregnated lens paper method and stored at -20°C or room temperature.
Main Results:
- The described procedure allows for the production of fresh frozen bone sections within minutes of tissue excision.
- The method yields high-quality sections suitable for various immunohistochemical analyses.
- Optimized cryostat settings and embedding techniques ensure tissue integrity and section completeness.
Conclusions:
- This novel technique provides a rapid and effective means for preparing fresh frozen bone biopsies.
- The procedure enhances the utility of bone biopsies for immunohistochemical studies in research and diagnostics.
- The method is adaptable for a range of immunohistochemical applications, improving workflow efficiency.