Tissue preparation process, embedding and block preparation in research area

Tissue preparation process, embedding and block preparation methodology followed in research area

1. TISSUE PREPARATION PROCESS, EMBEDDING AND BLOCK PREPARATION IN RESEARCH AREA

2. Tissue Preparation Tissue preparation process describes the steps required to take animal or human tissue from fixation to the state where it is completely infiltrated with a suitable histological wax and can be embedded ready for section cutting on the microtome. Tissue processing can be performed- Manually (hand processing), Automated tissue processing machine (a “tissue processor”).

3. Modern enclosed tissue processor

4. Tissue processing flow chart

5. 1. Obtaining a fresh specimen Fresh tissue specimens will come from various sources. It is important that they are handled carefully and appropriately fixed as soon as possible after dissection. Immediate fixation of the sample is recommended to prevent any damage.

6. 2. Fixation Hardening of the organ in such a way that its original form is retained and it’s constituents do not spread out while cutting. Small block of tissue is immersed in neutral buffered formalin 10%. The fixative should be 10-15 times more in volume than the specimen.

7. Commonly used Fixatives 10% Formalin Solution. Buffered Neutral formalin solution. Formalin Alcohol [Glycogen, Good Plasmatic Fixation]. Acetone [Rabbies]. Ethyl Alcohol. Zinker Fluid [Bone marrow aspirants]. Heat [5-10mm thick piece of tissue, boil in 0.9% of NaCl solution for 2-3 mins.] Note: Generally the specimen should fix for between 6 and 24 hours.

8. 3. Tissue processing Processing tissues into thin microscopic sections is usually done using a paraffin block, as follows: Dehydration Clearing Embedding

9. Dehydration: It is the first step, which involves immersing your specimen in increasing concentrations of alcohol to remove the water and formalin from the tissue. Clearing: It is the next step, in which an organic solvent such as xylene is used to remove the alcohol and allow infiltration with paraffin wax. Embedding: It is the final step, where specimens are infiltrated with the embedding agent – usually paraffin wax. The tissue becomes surrounded by a large block of molten paraffin wax, creating what is now referred to as the “block”. Once the block solidifies, it provides a support matrix that allows very thin sectioning.

10. 4. Sectioning The tissue specimens are cut into sections that can be placed on a slide. Blocks are chilled on a refrigerated plate or ice tray for 10 minutes before sectioning. A microtome is used to slice extremely thin tissue sections off the block in the form of a ribbon. The microtome can be pre-set to cut at different thicknesses, but most tissues are cut at around 5 µm.

11. Once cut, the tissue ribbons are carefully transferred to a warm water bath. Here they are allowed to float on the surface, and can then be scooped up onto a slide placed under the water level. Charged slides work best for this process – they improve tissue adhesion to the glass, and help to reduce the chance of sections washing off the slide during staining. Slides should be clearly labelled, and then allowed to dry upright at 37 o C for a few hours to gently melt the excess paraffin wax, leaving the tissue section intact.

12. 5. Staining Most cells are transparent, and appear almost colourless when unstained. Histochemical stains (typically haematoxylin and eosin) are therefore used to provide contrast to tissue sections, making tissue structures more visible and easier to evaluate. Following staining, a cover slip is mounted over the tissue specimen on the slide, using optical grade glue, to help protect the specimen.

13. Embedding / Blocking Embedding / Blocking is the final step, where specimens are infiltrated with the embedding agent – usually paraffin wax. The tissue becomes surrounded by a large block of molten paraffin wax, creating what is now referred to as the “block”. Once the block solidifies, it provides a support matrix that allows very thin sectioning.

14. Why is Tissue Preparation Important?? This is useful in establishing pathogenesis and pathology of any disease caused by Bacteria, Virus, mycoplasma, t oxin, parasite, poison, etc. There are certain diseases which histopathological examinations of tissues is the only alternative to diagnose the disease eg: Bovine Spongiform encephalopathy. In some cases the tissues of dead animals are only available material for diagnosis.

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