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 37oC 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, toxin, 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.

15. Thank you