Notes on staining techniques

Haematoxylin and eosin (H&E)

• This is the most commonly used stain in histology and pathology. Haematoxylin is a basic dye which stains acidic structures a purplish-blue. In contrast, eosin is an acidic dye which stains basic structures pinkish-red. In the mammalian cell, most of the acidic structures reside in the nucleus due to the presence of nuclear DNA, whereas most of the cytoplasmic structures are basic. Thus, nuclei stain purplish-blue and the cytoplasm a pinkish-red. If a cell’s cytoplasm contains abundant RNA, then the cytoplasm may have a purplish tint. In the first micrograph, the serous cells of the parotid gland show dense granular purple cytoplasm which is full of zymogen granules.

• The intensity of the staining by H&E depends upon many factors including tissue processing and fixation, the thickness of the tissue section and the formulation of the stain. The thickness of the section has the most impact upon the staining intensity as can be seen in some thicker, low-power images throughout this text.

• The second micrograph shows early secretory endometrium with subnuclear vacuolation in the glands which are easily demarcated from the background endometrial stroma.

Periodic acid–Schiff (PAS)

• The periodic acid–Schiff (PAS) reaction demonstrates the presence of complex carbohydrates by staining them a deep magenta colour. This is referred to as ‘ PAS positive’ . PAS will stain pure complex carbohydrates, such as glycogen in hepatocytes and muscle cells, but will also stain carbohydrates when they are bound to lipids and proteins. Basement membranes and the brush borders of kidney tubules and the small and large intestines are PAS positive, as is cartilage and some collagen.

Diastase periodic acid–Schiff (D-PAS, PASD, PASF)

• Many of these complex carbohydrate–lipid or protein compounds are present in basement membranes, fungal cell walls and some neutral mucin. In order to differentiate between pure glycogen and these other compounds, glycogen can be eradicated in the tissue section by pre-treatment with the enzyme diastase (amylase) prior to the PAS reaction.

Alcian blue (acid mucin)

• Epithelial mucins secreted by epithelial cells are either acidic or neutral. Acidic mucins are present in goblet cells and oesophageal submucosal glands. Most invasive adenocarcinomas also produce acidic mucin. Alcian blue stains acidic mucin a vivid blue colour. In contrast, neutral mucins, e.g. those present in gastric foveolar cells and prostate glands, are PAS positive. Alcian blue can also be used in combination with other stains such as H&E or van Gieson.

Masson trichrome

• Masson trichrome is a connective tissue stain as it is used to demonstrate supporting tissue elements such as collagen. As the name implies, this staining method produces three colours in tissue sections. Nuclei and other basophilic structures are stained black, collagen is stained green or blue depending on which variant of the staining protocol is used, and muscle, erythrocytes and keratin are stained bright red. In pathology, it is used routinely in liver and kidney biopsies.

Martius Scarlet Blue (MSB)

• MSB is another connective tissue stain. This three-colour staining method stains fibrin red, erythrocytes yellow and collagen blue. It is also useful for the examination of thrombi in histological sections, particularly in the autopsy setting.

Miller’s elastic van Gieson (EVG)

• This stains collagen red and elastic fibres black. It is particularly useful in staining the elastic laminae of arteries (black), the smooth muscle of the media and erythrocytes (yellow) and collagenous tissue (red).

Phosphotungstic acid haematoxylin

• This stain demonstrates muscle cross-striations such as in the myocytes of the heart. It may also be used to demonstrate the presence of mitochondria in particular types of tumours.

Gordon & Sweet’s reticulin

• This is a silver-based stain that demonstrates reticulin fibres of supporting tissues which are stained blue/black by this technique. It is particularly useful in the interpretation of liver architecture (illustrated in the micrograph), demonstrating the liver cell plates. Background tissues may be counterstained with a red dye (neutral red).

Sirius red

• This stain is most commonly used to detect amyloid deposits. These are acidophilic (eosinophilic) deposits of abnormal protein which can occur in many different tissue sites. Amyloid is characterised by beta pleated sheets on electron microscopy. Congo red is a similar stain to Sirius red. Both stain amyloid red and display apple-green birefringence under polarised light.

Giemsa stain

• This stain is mainly used to stain smears of blood cells and other cells e.g. in bone marrow and in neuropathology. It can also be used to demonstrate some microorganisms. Nuclei are stained dark blue to violet, background cytoplasm pale blue and erythrocytes pale pink.

Gram stain

• This method is mainly used in smears of bacteria in diagnostic microbiology, but can also be used in histological sections to show whether any bacteria are present and, if so, whether they are Gram-positive or Gram-negative. Gram-positive bacteria stain dark blue as illustrated in the micrograph opposite.

Perls’ stain

• This stain is used to demonstrate the presence of ferric iron in tissues, usually at the site of old bleeding where the iron-containing pigment, haemosiderin (derived from local haemoglobin breakdown), accumulates. It can also be used to demonstrate accumulation of iron in various tissues in the primary iron storage disease haemochromatosis and can be used for the identification of ferruginous asbestos bodies in lung tissue sections.

Masson-Fontana

• This stain is used to demonstrate melanin pigment in tiss ues but can also identify argentaffin granules and some neurosecretory granules. This stain can be used in the diagnosis of malignant melanoma as well as some melanin-producing microorganisms, e.g. Cryptococcus . Melanin pigment stains black and the background tissue stains pink.

Wade–Fite (modified Ziehl–Neelsen stain)

• This stain is used for identification of Mycobacterium leprae and atypical Mycobacteria and is a modification of the Ziehl–Neelsen stain for Mycobacterium tuberculosis organisms which are more acid and alcohol fast. The organisms stain red whilst the background tissues stain blue.

Ziehl–Neelsen

• This stain can be used to demonstrate Mycobacterium tuberculosis . This bacterium possesses a protective capsule containing lipids that affects the rate at which dyes move into and out of the organism during staining. Two dyes are used, basic fuchsin mixed with phenol and methylene blue. The first dye (which is red) is forced into all the tissues in the section (including any Mycobacteria ) by heating. The section is then exposed to acid and alcohol which wash the red dye out of everything except the Mycobacteria , which hold on to it because of the lipid capsule. All the other tissues are free to take up the contrasting blue dye, but the Mycobacteria remain red against a blue background.

Goldner’s trichrome stain

• This method is applied to acrylic resin sections of undecalcified bone to distinguish between mineralised bone and unmineralised osteoid. It has a haematoxylin component which also stains the nuclei of osteoblasts, osteocytes, osteoclasts and marrow cells.

• The von Kossa silver method stains calcium deposits black. It can be used in a variety of scenarios, including distinguishing between mineralised bone and osteoid and in identifying dystrophic calcium in tissues and in conditions such as pseudogout and malakoplakia.

Cresyl fast violet

• This is a basic dye commonly used in staining tissues of the brain and spinal cord. The neuropil stains purplish/blue and the Nissl substance of neurones stains dark blue due to the abundance of RNA. A modified version of this stain is also used to stain Helicobacter pylori organisms in gastric biopsies as illustrated in the micrograph.