12-11-14: Fixative Protocols and Recipes

from: http://www.ihcworld.com/_protocols/histology/fixatives.htm

Fixative Protocols and Recipes

Formalin Solution (10%, unbuffered):

Formaldehyde (37-40%) ------------- ----10 ml
Distilled water --------------------------- 90 ml
Mix well.

Formalin Solution (20%, unbuffered):

Formaldehyde (37-40%) ----------------- 20 ml
Distilled water --------------------------- 80 ml
Mix well.

Formalin Solution (10%, buffered neutral):

Formaldehyde (37-40%) ----------------- 100 ml
Distilled water --------------------------- 900 ml
NaH2PO4 ---------------------------------- 4.0 g
Na2HPO4 (anhydrous) ------------------- 6.5 g
Mix to dissolve.

Formalin Solution (20%, buffered neutral):

Formaldehyde (37-40%) ---------------- 200 ml
Distilled water -------------------------- 800 ml
NaH2PO4 --------------------------------- 4.0 g
Na2HPO4 (anhydrous) ------------------ 6.5 g
Mix to dissolve.

Formalin-Acetic Acid Solution:

Formaldehyde (37-40%) ---------------- 10 ml
Distilled water --------------------------- 90 ml
Glacial acetic acid ----------------------- 5 ml
Mix well.

Formalin-Alcoholic Solution:
Formaldehyde (37-40%) --------------- 10 ml
Ethanol (80%) -------------------------- 90 ml
Mix well.

Bouin Solution:
Picric acid (saturated) ------------------- 75 ml
Formaldehyde (37-40%) ------------------ 25 ml
Glacial acetic acid ------------------------- 5 ml
Mix well. For routine surgical material, especially for preserving soft and delicate structures such as
brain tissues.

Carnoy Solution:
Ethanol (absolute) ----------------------- 60 ml
Chloroform ------------------------------- 30 ml
Glacial acetic acid ----------------------- 10 ml
Mix well.
Note: Used for fixation of DNA, RNA, Nissl granules and glycogen.
Helly Solution:
Mercuric chloride ---------------------- 5 g
Potassium dichromate ---------------- 2.5 g
Distilled water ------------------------- 100 ml
Heat - cool - filter in brown bottle. Add 5 ml formaldehyde just before use. Wash in running
tap water for 24 hours before dehydration.
Note: Used for blood forming organs such as bone marrow, liver and spleen.

Susa Solution:
Stock Solution A:
Mercuric chloride ----------------------- 4.5 g
Sodium chloride ------------------------- 0.5 g
Trichloracetic acid ---------------------- 2 g
Distilled water --------------------------- 80 ml
Stock Solution B:
Glacial acetic acid ---------------------- 4 ml
Formaldehyde (37-40%) ---------------- 20 ml
Mix Solution A and B. For hard tissues such as inner ear with excellent penetration and little shrinkage.

Zenker Solution:
Mercuric chloride ---------------------- 5 g
Potassium dichromate ---------------- 2.5 g
Distilled water ------------------------- 100 ml
Heat - cool - filter in brown bottle. Add 5 ml of glacial acetic acid just before use. Wash fixed tissue
in running tap water for 24 hours before dehydration. Never use metal forceps or metal container
when handling tissues fixed in Zenker Solution.

Note: Used for bloody specimens such as spleen as well as connective tissues.

Zinc Fixative:
0.1M Tris Buffer, pH 7.4:
Tris Base ----------------------------- 12.1 g (TRIZMA)
1N HCL ------------------------------- 81.5 ml
Distilled water ----------------------- 900 ml

Zinc Fixative:
Calcium Acetate ---------------------- 0.5 g
Zinc Acetate --------------------------- 5.0 g
Zinc Chloride -------------------------- 5.0 g
0.1M Tris Buffer made above ------- 1000 ml
Mix to dissolve. The final pH will be approximately 6.5-7.0. Do not readjust the pH, as this will
cause the zinc to come out of solution. Store Zinc Fixative at room temperature. Fix tissues for 24
to 48 hours.

Davidson's Fixative Protocol *) siehe unten see below

4% Paraformaldehyde in 0.1M Phosphate Buffer

0.2M Phosphate Buffer (PB), pH7.4:

Na2HPO4 -------------------- 21.8 g
NaH2PO4 -------------------- 6.4 g
Distilled water ------------- 1000 ml

0.1M Phosphate Buffer, pH7.4:

0.2M PB --------------------- 500 ml
Distilled water -------------- 500 ml

4% Paraformaldehyde in 0.1M Phosphate Buffer:

Paraformaldehyde ------------------- 40 g
 0.1M Phosphate buffer ------------ 1000 ml

Heat to 60-65 ºC while stirring. Add a few drops of 1N NaOH until solution clear. Continue to stir to
dissolve. Cool the solution and filter.

Note: This solution is often used for animal perfusion.

4% Paraformaldehyde-1% Glutaraldehyde in 0.1M Phosphate Buffer

0.2M Phosphate Buffer (PB), pH7.4:

Na2HPO4 -------------------- 21.8 g
NaH2PO4 -------------------- 6.4 g
Distilled water ------------- 1000 ml

0.1M Phosphate Buffer, pH7.4:

0.2M PB ---------------------- 500 ml
Distilled water -------------- 500 ml

4% Paraformaldehyde-1% Glutaraldehyde in 0.1M PB:

Paraformaldehyde --------------- 40 g
0.1M Phosphate buffer -------- 1000 ml

Heat to 60-65 ºC while stirring. Add a few drops of 1N NaOH until solution clear. Continue to stir to
dissolve. Cool the solution and filter. Add 20 ml of 50% glutaraldehyde and mix well.

Note: This solution is often used for animal perfusion, especially for electron microscopy.
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Davidson's Fixative Protocol
http://www.ihcworld.com/_protocols/histology/davidson_fixative.htm

Davidson's Fixative Protocol

Robert S. Richmond, M.D., F.C.A.P.

Pathologist, USA

Principle: Davidson's (sometimes called Hartmann's) fixative is a rapid fixative which gives good
nuclear detail with minimal formalin pigment. Specimens cut and stain as if fixed in ordinary formalin.
Davidson's fixative contains no mercury or other metals.

Specimen: Davidson's fixative is particularly useful for preparing tumors, bone marrow specimens,
gynecologic material, fatty breast, and medical biopsies. It may be used for testicular biopsies and for
overnight fixation of fat containing lymph nodes. Fixation of small specimens is rapid. Exposure to the
fixative should be limited to 24 hours (tissue may be transferred to neutral buffered formalin or 70% alcohol
for storage).

Procedure: Mix [37% FA-Alcohol-Acetic Acid-Tap water]

strong formalin (37%): 2 parts or 500 mL

alcohol: 3 parts or 750 mL
glacial acetic acid: 1 part or 250 mL
tap water: 3 parts or 750 mL
eosin: enough to color

Reagents and Supplies:

Any source of these chemicals should be satisfactory. Proportions are not very critical. Reagent alcohol, or
waste alcohol from staining, may be used.

Quality Control:

Davidson's fixative is clear and has no color other than that imparted by eosin. Color should be sufficient to render identification
easy. The odor changes with age as esterification produces a characteristic "airplane dope" odor of ethyl acetate. Tissues placed in
Davidson's fixative rapidly turn white and opaque, while blood turns brown.

Safety and Environmental:

Acetic acid is quite corrosive. Davidson's fixative may be stored at room temperature in plastic or glass
containers for an indefinite time.

Note:

Here is another similar recipe for Davidson's Fixative from histonet and it is recommended for eye fixation.

References:

Davidson's fixative is not mentioned in most standard reference works on histology.

It is named after William McKay Davidson, a British hematologist, and was publicized by Moore and Barr
in their well-known studies of sex chromatin. Davidson's fixative is often called Hartmann's fixative
because of its introduction by Dr. William H. Hartmann (later at Vanderbilt) while he was at Johns Hopkins.

1. Moore KL, Graham MA, and Barr ML. "The detection of chromosomal sex in hermaphrodites from a skin
biopsy." Surgery, Gynecology and Obstetrics 1953;96:641-648.

2. Moore KL and Barr ML. "Nuclear morphology, according to sex, in human tissues." Acta Anatomica 1954;21:197-208.

3. Procedure written by Robert S. Richmond, M.D., F.C.A.P., July 1997