Fixatives for Optical Microscopy

Formalin breaks down to produce formic acid, which may affect some types of tissue. Formalin Neutral Buffer Solution is made by adding sodium phosphate to formalin to adjust the pH to approximately 7.4; there is no need to worry about the effects of acid on the tissues.

Formalin Neutral Buffer Solution is not only excellent as a routine fixative but also can be used for special purposes such as histochemical methods (including staining of enzymes and hemoglobin) and staining of tissues for which common formalin solutions are not suitable (tissues containing viscous polysaccharides, glycogen, or neurofibrils). This solution can also be used for tissue fixation for electron microscopy.

Product Name	Composition	Formaldehyde content	pH	Penetration
10% Formalin Neutral Buffer Solution	・Formalin (undiluted): 100 mL ・Monosodium phosphate dihydrate: 4.5 g ・Disodium phosphate anhydrous: 6.5 g Add ion exchanged water to make 1 L.	3.8～4.1 w/w％	7.4～7.5	・Good in order of 20% > 15% > 10% ・10% is suitable if the tissue is to be kept in the fixative for more than several days. ・20% is suitable for 1–2 days of fixation. ・15% falls between the above two concentrations.
15% Formalin Neutral Buffer Solution	・Formalin (undiluted): 150 mL ・Monosodium phosphate dihydrate: 4.5 g ・Disodium phosphate anhydrous: 6.5 g Add ion exchanged water to make 1 L.	5.8～6.1 w/w％	7.35～7.45
20% Formalin Neutral Buffer Solution	・Formalin (undiluted): 200 mL ・Monosodium phosphate dihydrate: 4.5 g ・Disodium phosphate anhydrous: 6.5 g Add ion exchanged water to make 1 L.	7.6～8.2 w/w％	7.3～7.5

This product is an odorless fixative made by adding wine extract, which lessens the irritating and unpleasant odor of formalin, to neutral buffered formalin solution (Lilly’s formalin). For safety reasons, the odor is not completely masked so that it can be recognized as formalin.

Features

• Ideal for tissue fixation for immunohistochemical staining using the enzyme-labeled antibody method.
• Odor-lessening component is stable without losing its effect, even in tissues that have undergone considerable autolysis.
• Stable at room temperature for more than 2 years.
• Frees operating rooms and pathology laboratories from the unpleasant odor of formalin–biopsy materials, tissues resected for examination, surgical resection materials, and tissues obtained in autopsies can all be fixed immediately.
• Tissue penetration and fixation are equivalent or superior to neutral buffered formalin solution.

Example of staining using Mildform

• N: Neutral. Mildform is produced based on neutral buffered formalin solution.
• M: methanol is added to the formulation. The addition of methanol enhances tissue penetration and fixation. The concentration of methanol is 10%.
• The NM type is especially suitable for quick, short-term fixation. This product is also an excellent formalin/methanol (FM) fixative suitable for the fixation of specimens containing a large amount of connective tissue or adipose tissue.

Product Name	Composition	Features
Carnoy Solution	・Pure ethanol: 600 mL ・Chloroform: 300 mL ・Glacial acetic acid: 100 mL	Superior in penetrability to absolute alcohol, and suitable for the fixation of glycogen, nucleic acid, etc. For tissue specimens 3–4 mm in size, fixation is completed in 2–4 hours. After fixation, the tissue is transferred to pure alcohol to remove chloroform and acetic acid, and is then embedded. The strong dehydrating/defatting effect of this fixative may cause substantial shrinkage and hardening of tissues or cells, especially when the fixation time is long.
Bouin Solution	・Saturated picric acid solution: 750 mL ・Formalin (undiluted): 250 mL ・Glacial acetic acid: 50 mL	Strong penetration; for small pieces of tissue, fixation is completed in 1–2 hours. The weak decalcifying effect of this fixative allows thin sections of fetal bone tissue to be made without undergoing a decalcification process. Regularly used in the fixation of endocrine tissues, glycogen, polysaccharides, etc. After fixation, tissue must be washed thoroughly with 70% ethanol until the solution ceases to be yellow in color.
Zamboni Solution	・Saturated picric acid solution: 150 mL ・Paraformaldehyde: 20 g Add phosphate buffer solution to make 1 L.	Mainly used as a fixative for immunohistochemical staining and electron microscopy. Suitable for the fixation of polypeptide antigens. Fixation should be done at 4 °C for 4–12 h. After fixation, tissue must be washed thoroughly with 70% ethanol until the solution ceases to be yellow in color.
4% Paraformaldehyde Phosphate Buffer Solution	・Paraformaldehyde: 20 g ・0.1 mol/L Phosphate buffer solution (pH 7.4): 500 mL	Mainly used as a fixative for immunohistochemical staining and electron microscopy. Suitable for the fixation of protein antigens. Fixation should be done at 4 °C for 4–12 h.
PLP Solution Set	・Solution A (lysine-phosphate buffer solution): 50 mL ・Solution B (10% paraformaldehyde solution): 50 mL ・Solution C (sodium metaperiodate solution): 50 mL	Neutral buffered fixative specialized for immunohistochemical staining. Suitable for the fixation of glycoprotein antigens. Used to detect complement or lymphocyte surface markers, which can easily lose their antigenicity.

Example of staining