Basic guide to histological staining and tissue preparation
Dr A McLeod

The typical thickness of a histological specimin is only 3 micrometers. At this thickness virtually all tissue structures are near-transparent. At the basic level a stain makes the specimin easier to see and because a stain affects different areas (e.g. nucleus vs cytoplasm) differently, a stain makes selected elements easier to distinguish. 

Choice of stain will depend on the tissue and the aim of the examination as well as the way the specimen has been prepared.

Preparing a tissue for staining
This process is generally automated and done overnight. It is needed to provide a sample sturdy enough for obtaining a thin section for the slide - it can however produce changes to the tissue called artifacts - things that are only there due to what we have done to the tissue and which don't mean anything diagnostically. The skilled pathologist can recognise these artifacts for what they are.

Once the wax block is finished, sections will be cut from it. This is usually done by hand and is a skilled job with the lab personnel making choices about which parts of the spcimen to place on slides.A number of slides will usually be prepared from different levels throughout the block. After this the thin section is stained and placed on s slide with a protective cover slip. For common stains and automatic process is normally used but rarely used stains are often done by hand.

In the UK, tissue and sections taken for histology are retained for att least 25 years.

Commonly used stains (alphabetically)

Giemsa: a mixture of methyline blue and eosin - the appearance is unsurprisingly similar to, but not identical to the appearance under H&E and Wrights stain. It also binds to some pathogenic organisms including plasmodium (causative of malaria), spirochaetes (syphillis), and trypanosomes (Chagas disease and sleeping sickness).

Haematoxylin and Eosin. A quick to apply and cheap stain that is the most-used preparation in most pathology labs.
Haematoxylin has a deep blue-purple color and stains nucleic acids by a complex, incompletely understood reaction. It stains the cell nucleus well enough to distinguish varying cell-type- and cancer-type-specific patterns of condensation of heterochromatin (hematoxylin staining) that are diagnostically important.
Eosin is pink and stains proteins nonspecifically. It stains, cytoplasm, extracellular matrix and nuceoli.
In a typical tissue, nuclei are stained blue, whereas the cytoplasm and extracellular matrix have varying degrees of pink staining. A clear area of cytoplasm near the nucleus represents the Golgi zone.

Oil red O:
This is used as a stain for neutral lipids. It is more soluble in fat than in either alcohol or water. It is useful in e.g. multiple sclerosis to detect the lipid-rich debris in macrophages from myelin breakdown in the CNS.

Papanicolaou Stain
This histology stain is used mainly on cytological specimens. Cells in smear preparations can be stained with Pap staining. Gynecological smears (Pap smears), sputum, urine, cerebrospinal fluid, abdominal fluid, pleural fluid, synovial fluid, semminal fluid and fine needle aspiration samples can all be stained with a Pap stain. This staining technique involving five dyes in three solutions.
Periodic Acid-Schiff (PAS): This histology stain is particularly useful for staining glycogen and other carbohydrates, but is useful for many things. It is often used to show glomeruli, basement membranes, and glycogen in the liver. PAS stains glycogen, mucin, mucoprotein, and glycoproteins magenta. The nuclei will stain blue. Collagen will stain pink.

Wright's stain:
Methylene blue (blue dye) has a positive charge and stains acidic granules as well as RNA and DNA all of which have negative charges; Eosin (a red dye) has a negative charge and so stains some granules and hemoglobin which are basic with positive charges, a red colour.

Ziehl-Nielson stain is used to identify acid-fast organisms such as the mycobacteria responsible for tuberculosis and leprosy.

Antibody Stains
Antibodies can now be made to attach to specific molecules - they can hence be used to stain these molecules and make them visible allowing, for example a helper T cell and a ctytotoxic T cell to be differentiated, or discovering if a breast cancer has receptors for oestrogen (which affects it's vulnerability to treatment with Tamoxifen).

Stains applied to living tissue
Stains applied to living tissue are called Vital stains. These are further divided into supravital (introduced to living tissue that has been removed from the body, but before cessation of the chemical life of the cells) and intravital (absorbed by living cells after injection into the body) stains.

Click here if you wish to reproduce any information from this page.


H&E Staining
Introduction to Microscopy: Open University
Introduction to Histology: Open University
Other stains


Updated June 2016