Staining, Histochemistry and Histotechnology
(Frequently Asked Questions)
Dr. John A. Kiernan
Department of Anatomy
and Cell Biology
The University of Western Ontario
replace xylene AND alcohols
Is there a product that replaces xylene AND alcohols in the staining procedure? Can you use it before and after the actual staining is done?
t-butanol, dioxane and tetrahydrofuran are miscible with wax, water and resinous mounting media. Of these, only t-butanol (= tertiary butyl alcohol) is suitable for ordinary use. (The other two have such hazards as fire, toxicity and explosive peroxide formation.) t-butanol is often used in botanical microtechnique; it is quite a bit more expensive than alcohol or xylene. n-butyl alcohol mixes with wax and mounting media and is also partly miscible with water. It's good when you use easily extracted stains (methyl green-pyronine, for example), but has unpleasant vapour.
2-butoxyethanol (butyl cellosolve) also has the right miscibilities, and is quite cheap because it's used on a big scale industrially.
For microwave processing, isopropyl alcohol is sometimes recommended. However, this does not mix with wax. It has to leave the specimen by vaporizing (boiling) under reduced pressure. This can lead to considerable tissue damage unless the temperature and pressure are just right (Bosch et al 1996).
Some staining methods work well, though slowly, without removing the paraffin beforehand (Kiernan 1996), provided that there has been no melting or softening of the wax after mounting the sections on their slides.
Bosch,MMC; Walspaap,CH; Boon,ME (1996): Lessons from the experimental stage of the two-step vacuum-microwave
method for histoprocessing. Eur. J. Morphol. 34(2), 127-130.
Kiernan,JA (1996): Staining paraffin sections without prior removal of the wax. Biotechnic & Histochemistry 71(6), 304-310.
John A. Kiernan
We use 99% isopropyl alcohol (IPA) instead ethanol AND xylene AFTER staining. It is especially useful after staining of lymph
nodes with a modified Maximov-Giemsa method. My laboratory has used
this modification more then 5 years and I have never seen the same excellent result in comparison with atlases of lymph nodes biopsy. Moreover, we use IPA with addition of a small amount of detergent for dehydration of samples. Four changes of 99% IPA+detergent is all you need between water and paraffin. We never have have problems with any tissues, including large samples of skin. Our HTs adore IPA.
Dr Yuri Krivolapov
Military Medical Academy