From Biopsy to Microscopy - Tissue processing for light microscopy

Summary

This video provides an insightful overview of the process involved in preparing tissue samples for light microscopy. It covers key stages such as fixation, where tissue is preserved; processing, which involves dehydration and clearing; embedding the tissue in paraffin wax; section cutting with a rotary microtome; and staining with Hematoxylin and Eosin to highlight cellular structures. The final step is mounting, where the tissue is sealed under a cover slip for permanent preservation. This comprehensive process ensures that tissue samples are accurately prepared for detailed microscopic examination.

Takeaways

• 😀 Fixation is crucial to preserve tissue and prevent decay.
• 😀 Tissues are processed by replacing water with molten paraffin wax.
• 😀 Alcohol solutions gradually dehydrate tissue to prevent shrinkage during processing.
• 😀 Clearing agents make tissue translucent and remove the remaining alcohol.
• 😀 Molten paraffin wax is used to block and support tissue for sectioning.
• 😀 Tissue blocks are sectioned with a rotary microtome to produce thin slices.
• 😀 Sections are floated on a warm water bath to remove wrinkles before mounting.
• 😀 Dewaxing and rehydrating bring sections back to water for staining.
• 😀 Hematoxylin and eosin are the primary stains used in histology.
• 😀 Hematoxylin stains nuclei blue to black, while eosin stains cytoplasm and intercellular components pink.
• 😀 The final step involves mounting the stained tissue sections with a clear mounting medium (PX) to preserve and display them.

Q & A

What is the purpose of fixation in tissue preparation for light microscopy?

Fixation preserves the tissue by preventing decay, ensuring that it remains intact for further processing and examination under the microscope.

Why is it necessary to replace the water in the tissue with paraffin wax during processing?

Water must be replaced with paraffin wax because paraffin is not soluble in alcohol, and it provides a solid medium to support the tissue, which helps in slicing thin sections for microscopy.

How does the clearing agent assist in tissue preparation?

Clearing agents, such as xylene, make the tissue translucent and remove the alcohol, enabling the tissue to be compatible with paraffin wax during the embedding process.

What is the role of a rotary microtome in tissue preparation?

A rotary microtome is used to slice the paraffin-embedded tissue into thin sections, typically around 5 microns thick, to prepare them for staining and microscopic examination.

Why are tissue sections stained with Hematoxylin and Eosin?

Hematoxylin and Eosin are used to stain tissue sections because Hematoxylin stains the nuclei blue to black, while Eosin stains the cytoplasm and intercellular components in pink, helping to differentiate the structures under the microscope.

What is the process of differentiation in staining, and why is it necessary?

Differentiation involves selectively removing excess Hematoxylin with a 1% hydrochloric acid solution to ensure that the nuclei are clearly visible without being overly stained.

Why is it important to use a non-toxic solvent like Holine in the clearing process?

Non-toxic solvents like Holine are safer for laboratory workers and still effectively clear the tissue to make it translucent, ensuring that the tissue is ready for embedding and sectioning.

How does the use of paraffin wax aid in tissue sectioning?

Paraffin wax provides a solid support structure that stabilizes the tissue, making it easier to cut into thin sections without distortion or tearing.

What is the purpose of mounting the stained tissue section with PX?

PX, a synthetic mounting medium, is used to mount the stained tissue sections by adhering a glass cover slip to the slide, ensuring the tissue remains in place while also having the same refractive index as glass to avoid distortion of light.

What does the term 'bringing the sections to water' refer to in the tissue processing procedure?

'Bringing the sections to water' refers to the process of rehydrating the tissue sections after dewaxing to remove the paraffin wax and make the sections compatible for staining with water-based dyes.