Hi Alida, this is a really good question. I think that the basis for this is the combination of basic and acidic dyes to stain acidic/basophilic, and basic/acidophilic molecules in tissue, respectively. I realize that your question is really asking why some stains won't work without their counterpart. I know that you must use basic dyes before acidic dyes, and alcohol before aqueous, otherwise staining will not work. I suggest Animal tissue techniques (A Series of books in biology) 3rd ed. by Gretchen L Humason. It was published in 1972, but is still one of the best histology books I've ever seen. You can get it on amazon for about $10, ISBN-10: 9780716706922 .

Hi Alida, this is a really good question. I think that the basis for this is the combination of basic and acidic dyes to stain acidic/basophilic, and basic/acidophilic molecules in tissue, respectively. I realize that your question is really asking why some stains won't work without their counterpart. I know that you must use basic dyes before acidic dyes, and alcohol before aqueous, otherwise staining will not work. I suggest Animal tissue techniques (A Series of books in biology) 3rd ed. by Gretchen L Humason. It was published in 1972, but is still one of the best histology books I've ever seen. You can get it on amazon for about $10, ISBN-10: 9780716706922 .

Dear Susan, thank you so much for this response, I will get this book right away! All the best

Alida

There is an updated 5th edition, but I haven't seen it because it was done by different authors, but you might consider it as well. Available from various sellers. https://www.addall.com/ is the best as finding stuff like this and comparing prices.

Thanks for the suggestion Matthew. There are a couple of copies available to ship from France. ~$20 total.

Look into: Romeis B (1968) Mikroskopische Technik. R. Muenchen, Wien: Oldenbourg Verlag. If you work in Neuroscience : Adams CWM (1965) Neurohistochemistry. Elsevier Amsterdam, if working in cytology: Boon ME, Drijver JS (1986) Routine cytological staining techniques: theoretical background and practice. Hampshire and London: Macmillan Education Ltd. If working with microwave techniques, Kok and Boon Microwave cookbook for microscopists. Coulomb Press, Leyden, 1992.

Hi Alida, I suggest you to refer the book CONN'S Biological stains: A Hand book of Dyes, Stains and Fluorochromes for Use in Biology and Medicine, edited by R.W. Horobin and J.A.Kiernan, A Biological Stain Commission Publication, 2002 (10th Edition). This book presents details of history, nomenclature, classification of biological stains, applications and mechanisms of staining, vital staining counterstaing and multiple staining, use of dyes, fluorohromes as indicators, in a chapterwise manner. Very informative book I referred. I am sure you get answers for all your questions regarding histological stains and staining from this book.

I am not used to histology so I cannot suggest you a reference book.

As far as I know, the main reason for counterstain is to give contrast to the image itself and increase the stain precision. Anyway, there might be chemical reasons for that. As an example, I used a stain protocol for collagen that used two stains. A first stain was used to stain unselectively everything, especially what might look like collagen or have similar charges. Then a second polyacid molecule selectively intercalate the triple helix displacing the first stain. Finally, the second stain was added and displace this polyacid molecule, staining collagen with a different color. In that case, the two stain were used so that the second stain would have been more selective for that specific target, avoiding to stain other portion of the sample.

I am not sure if this might help you or not.