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Metallic Fixatives
Metallic Fixatives
Working solution: (prepare immediately before use) 5. Susa-fixed tissues may be transferred directly to
B-5 stock solution: ---------- 20 ml 95% alcohol or absolute alcohol, thereby reducing
40% formaldehyde: ---------- 2 ml processing time.
Disadvantages:
Fixation time: 4 – 8 hours 1. Prolonged fixation of thick materials may produce
considerable shrinkage, hardening and bleaching;
Practical Applications: hence, tissues should not be more than 1 cm. thick.
1. Despite its mercuric chloride content and 2. RBC preservation is poor.
consequent problems with disposal, this solution is 3. Some cytoplasmic granules are dissolved.
popular for fixation of hematopoietic, bone marrow 4. Mercuric chloride deposits tend to form on tissues;
biopsies and lymphoid tissue. these may be removed by immersion of tissues in
2. Rapid fixation can be achieved in 1 1/2 - 2 hours. alcoholic iodine solution.
3. It produces excellent nuclear detail, provides good 5. Weigert's method of staining elastic fibers is not
results with many special stains, and is recommended possible in Susa fixed tissues.
for immunohisto-chemical staining. Precautions:
4. Sections will require the removal of mercury After using Heidenhain's Susa fixative, the tissue
pigment prior to staining. should be transferred directly to a high-grade alcohol,
5. Tissue should not be stored in this fixative but e.g. 96% or absolute alcohol, to avoid undue swelling
placed in 70% ethanol instead. of tissues caused by treatment with low-grade alcohol
6. Over-fixation hardens the tissue and makes cutting or water.
difficult.
7. The two working solutions are kept separate, since OXIZING FIXATIVES (OSMIC ACID;
the mixture is unstable. Mix just prior to use.
Os04)
8. Some B-5 solutions will form precipitate on
• Traditionally used in electron microscopy both
standing, but this is of no consequence.
as a fixative and a heavy metal stain
9. As with all mercuric chloride fixatives, the mercury
• excellent stain for lipids in membranous
pigment can be removed by de-zenkerization.
structures and vesicles
• should be kept in DARK COLORED, chemically
Heidenhain's Susa Solution
clean bottle to prevent evaporation
Is recommended mainly for tumor biopsies especially
andreduction by sunlight and organic matter
of the skin; it is an excellent cytologic fixative.
• inhibits hematoxylin and makes
FORMULA: counterstaining difficult produces
Mercuric chloride-----------45 gm > Prevention addition of saturated Mercuric Chloride
Sodium chloride------------- 5 gm > Remedy: Black osmic oxide crystals may be
Trichloroacetic acid-------- 20 gm dissolved in cold water
Glacial acetic---------------- 40 ml
Acid Formaldehyde 40%----- 200 ml
Procedure:
40% Distilled water----------- 800 ml
1. Wash the sample four times with PBS.
2. Fix with 2% Paraformaldehyde, 0.1%
Fixation time: 3-12 hours Glutaraldehyde in PBS for 30 minutes.
3. Wash four times with PBS.
4. Wash four times with double distilled water.
Advantages: 5. Prepare 0.1% OsO4 solution by diluting the 4%
stock solution in double distilled water.
1. It penetrates and fixes tissues rapidly and evenly. 6. Incubate the sample with 0.1% OsO4 for 30
2. It produces minimum shrinkage and hardening of minutes.
tissues due to the counter-balance of the swelling 7. Wash four times with double distilled water.
effects of acids and the shrinkage effect of mercury.
3. It permits most staining procedures to be done,
Flemming's Solution
including silver impregnation, producing brilliant
is the most common chrome-osmium acetic acid
results with sharp nuclear and cytoplasmic details.
fixative used, recommended for nuclear preparation
4. It permits easier sectioning of large blocks of
of such sections.
fibrous connective tissues.
TRICHLOROACETIC ACID
➢ Trichloroacetic Acid (TCA) is a reagent that is
used for the precipitation of proteins and
nucleic acids. It is also used as a decalcifier
and fixative in microscopy.
➢ Addition of TCA to a final concentration of
10% (w/v) will precipitate most proteins from
solution. The excess TCA can be removed
from protein pellets by washes with buffer.
Advantages:
1. It precipitates proteins.
2. Its marked swelling effect on tissues serves to
counteract shrinkage produced by other fixatives.
3. It may be used as a weak decalcifying agent.
4. Its softening effect on dense fibrous tissues
facilitates preparation of such sections.
ACETONE
➢ Acetone is not recommended as
morphological fixative for tissue blocks,
mainly because of its shrinkage and poor
preservation effects. Its use is reserved for the
fixation of cryostat sections or for tissues in
which enzymes have to be preserved.
➢ It is used to fix specimens at cold
temperatures (0 to 4°C). Fixation time may
vary from several minutes (for cell smears,
cryostat sections) to several hours (1-24 hours
for small tissue blocks).