Outline the steps from dissection of the tissue to producing a section
on a slide as provided for this practical and as would be carried out in a
Specimens infiltrated with a liquid agent that has been
converted into a solid with suitable physical properties allowing these sections to
be sliced from it. Paraffin wax-based histological waxes are most popular for
light microscopy. Paraffin-sections are prepared in the ‘rotary microtone’. The
next step include a careful examination and specimen description including
appearance, dimensions etc. These are placed into small perforated baskets and
loaded into a tissue processor for processing through wax. Tissue processors
allow the specimens to be infiltrated with a sequence of different solvents
finishing in molten wax. Processed specimens are kept in an embedding center. Carefully
orientated specimens determining the plane through which the section will be
cut are placed in wax-filled moulds. Further wax is added to form a block that
later cools down to solidify. Blocks are firmly clamped in the specimen holder
is a microtome and thin single layer of cell sections are cut off. Sections are
floated out on the surface of warm water in a floatation bath in order to
flatten them and are picked up onto microscope slides. These are dried and
Why are the majority of samples sent to the histopathology laboratory
as ‘fixed’ specimens?
Specimens are usually sent to a histopathology
laboratory fixed to prevent drying-out of the specimen or degeneration. Most
poplar fixing agent is formaldehyde usually in the form of a phosphate buffer
solution. Ideally the sample should be immersed 6-12 hrs before being processed
with a generous volume of (fixative) formalin compared to the size of specimen.
Fixing a fresh specimen as soon as possible reduces the risk of inaccuracies.
Fixation prevents decay, preserving the specimen cells and tissue in a ‘life
like’ state by stopping enzyme activity killing microorganisms. Hardening
specimen while maintained sufficient molecular structure will enable suitable
staining methods to be applied. The sooner fixation Initiated following
separation a specimen from its blood supply, the better the results will be.
Was direct or indirect labelling used in this practical and how did you
decide this from the protocol?
Direct labelling uses a single antibody against the
target whereas indirect labelling used two antibody. In this case direct
labelling was used
What are the main differences between the detection of a protein by a
monoclonal antibody and a polyclonal antiserum?
Polyclonal antibodies are antibodies that are secreted
by different B cell lineages within the body. Monoclonal antibody comes from
single cell lineage. They are a collection of immunoglobin molecules that react
against a specific antigen, each identifying a different epitope. Polyclonal
antibodies are made using several different immune cells. They will have
affinity for the same antigens but different epitopes. Monoclonal antibodies
bind only a single epitope while polyclonal antibodies bind to different
epitopes in the same protein.
Why might antigen retrieval be needed in immunocytochemistry and what are the main methods used?
Fixation for preservation of tissue morphology sometimes
can have a negative impact on detecting IHC/ICC or it can alter protein
biochemistry so that the epitope can no longer bin to the primary antibody.
Antigen retrieval includes techniques that reverse this masking effect of an epitope.
This can be done using simple methods such as changing pH or altering the
cation concentration of the antibody which will in turn affect the affinity of
an antibody to its epitope. Two main techniques of antigen retrieval used are
protease-induced epitope retrieval (PIER) and heat-induced epitope retrieval
(HIER). In PIER antibody-epitome binding
is restored using enzymes such as proteinase K, Trypsin, and Pepsin. However
this method has a low success rate in restoring immunoreactivity. In HIER some
cross-links and restores secondary or tertiary structure of the epitope and is
usually carried out using microwave ovens, pressure cookers, vegetable steamers
or water baths. HIER has a higher success rate than PIER. HIER is temperature,
buffer and pH sensitive.
How is the intensity of staining scored in a histopathology lab? How
can this be standardized to allow comparison between different patients and
The H-score is calculated using a semi-quantitative
approach which calculates a summation of the percentage of the area stained at
each intensity level multiplied by the weighed intensity. The formula used to
calculate H-score is 1 × (% cells 1+) + 2 × (% cells 2+) + 3 × (% cells 3+).
In this practical a control slide was not used. In a hospital lab what
controls might you consider using and what is the importance of control slides?
It is important to ensure that a control is used because
staining a tissue that is not known to express the point of interest will allow
to compare specific binding to the antibody. Co troll will allow to achieve
staining efficiency. Both primary and secondary antibodies need a level of
optimization while comparing IHC to false positives due to over saturation.
Using an isotype control helps to confirm the primary antibody is not binding
to the immunoglobulin of the tissue but the specific antigen.