1. Dr Vivien Rolfe De Montfort University This is an Open Educational Resource (OER) that is globally available on the web Creative Commons BY SA

2.  Histology is the microscopic study of normal tissue  Histopathology is the microscopic study of diseased tissue  Cytology is the………………………..  Cytopathology is the…………………….

3.  The light microscope is used to examine histology slides. You should be familiar with the parts, operation and principles of the microscope.  Use our microscopy OERs to learn how to use the equipment:  https://www.youtube.com/watch?v=NJot RNdt3JM

4. To visualise cell structures and the chemical nature of tissues.

5. By Dey on Flickr CC BY NC SA

6.  A dye is a coloured compound that binds to a substrate.  It comprises of the chromogen (colour) and auxochrome (substrate binding component). Chromogen Auxochrome Substrate

7.  Dyes are arranged in 16 groups: Basic dyes – coloured cations bind to anionic substrate e.g. DNA Acid dyes – coloured anions bind to cationic substrate e.g. cytoplasm Mordant dyes – conjugated to metal salts. Direct dyes Reactive dyes Others (Kiernan 2007, Histological and Histochemical Methods).

8. The most widely used stain around the world. Known as the “international stain”. A simple histological stain with two colours.

9. Haematoxylin From the bark of the logwood tree Haematoxylin From the bark of the logwood tree Haematoxylum campechianum http://farm3.static.flickr.com/2511/3725895561_f7f7d1a749_o.jpg, Flickr, CC BY SA Kurt Stüber, Wikimedia Commons

10.  Most staining is a simple nuclear stain and a counterstain.  In most staining protocols, lowest molecular weight dyes are applied first (e.g. haematoxylin), differentiated, and then larger counterstains are applied.  “Haematoxylin” is a mordant (metal complexing) dye; the cationic / basic dye-metal complex binds to nucleic acids in DNA.

11.  Haematoxylin is extracted and oxidised to haematein. When it complexes with metal it is more rightly called haemalum.  It should rightly be the haemalum and eosin stain.  Aluminium-haematein (e.g. Mayer’s) is colourised in alkaline tap water (e.g. Scott’s) which is called blueing, simply to get the desired final colour.  Iron-haematein is a deeper blue-black and is differentiated in stronger acids (e.g. protocols that use Weigert’s haematoxylin).

12. Eosin Synthesised from fluorescein, a synthetic organic compound Used in cosmetics Fluorescein, Benjah-bmm27, Wikimedia Commons

13.  Anionic /acid countertain.  Eosin Y (a product of fluorescein) binds to amino acids and most cellular components, but not for example, larger molecular structures such as collagen which are also anionic.  Stains pink/red.

14.  Dewax, take sections to water  Apply haematoxylin  Rinse  Differentiate in acid alcohol (removes dye from cytoplasm leaving crisp nuclei)  Rinse  Blue  Rinse  Apply eosin (alcohol based dye)  Rinse in alcohol  Dehydrate and clear

15. Will vary on the tissue makeup. Cytoplasm-rich muscle will look very pink. Nuclei-dense tissues e.g. glands look more purple.

16.  Histological and Histochemical Methods. 4 th Edition. By JA Kiernan. 2007. Scion publishing. Available: http://www.scionpublishing.com  Histopathology: Fundamentals of Biomedical Science. By G Orchard and B Nation. 2012. Oxford University Press. Available: http://www.oup.com/uk/orc/bin/fbs/  Laboratory skills open educational resources. De Montfort University. Available: https://www.youtube.com/user/biologycourses  Haematoxylin and eosin videos and open educational resources. De Montfort University. Available: http://hlsweb.dmu.ac.uk/ahs/elearning/RITA/Resources.ht ml

17. Dr Vivien Rolfe De Montfort University This is an Open Educational Resource (OER) that is globally available on the web Creative Commons BY SA