Up Your Stain Game With These 7 Non-Fluorescent Histology Dyes

Histological stains that have an affinity for specific cellular components have been in use since at least the 1770s when John Hill used carmine to study tissues. Stain variety exploded during the 1800s with German dye manufacturers, such as BASF, developing aniline, methylene blue, and eosin. Eosin is still in use today with hematoxylin for H&E staining. 

Since the advent of immunofluorescence and fluorescent protein tagging, which provides very specific labeling, dyes have been relegated to only the most basic imaging. If you don’t need specific proteins labeled, dyes can be a cheap and useful alternative offering simple sample preparation with no or basic microscope techniques. Today we will discuss 7 dyes you should know for use in a laboratory setting.

Perls’ Prussian Blue Stain (1867)

Prussian blue staining is used to identify iron in histology sections and is one of the most common early stains that is still used in medicine today. In pathology, the stain is used to identify excess iron in a sample due to conditions such as blood disorders, lead poisoning, alcoholism, and splenectomy. Prussian blue is also used to label asbestosis fibers dark blue/black. 

Perls’ Prussian Blue is technically not a stain, but a chemical reaction. Right before staining,  hydrochloric acid and potassium ferrocyanide are mixed together. In the presence of free iron, potassium ferric ferrocyanide (Prussian Blue) is precipitated. Prussian Blue is highly colored and highly water-insoluble complex. 

 H&E Staining (1876)

H&E staining uses hematoxylin (haemotoxylin) and eosin to bind acidic and basic structures within the cell, respectively. Practically, this means tissue sections will appear to have purple nuclei and pink cytoplasm and membranes. H&E is the most common histology stain to create contrast in otherwise transparent tissues. H&E is still widely used in pathology labs but often overlooked for tissues of normal morphology. There is a good reason it has been in use for over 100 years and is the first histological stain new scientists learn–it works!

Congo Red Stain (1883)

Congo Red is an azo dye that was originally synthesized as a textile dye. Congo Red staining is used for the visualization of amyloid deposits. Interestingly, when Congo Red binds to amyloid structures, the dye will appear green with polarized microscopy due to a special birefringent property. Although Congo Red is often used with transmitted light microscopy (bright field or polarization), it can also be used for a fluorescent stain. Congo Red has an excitation maximum at 497nm and emission at 614nm. 

Other uses for Congo Red stain include the cell wall of plants and fungi, the outer membrane of gram-negative bacteria, and even tapeworm cysts. Glycoprotein rich structures in cells and organisms bind Congo Red and will stain positive. Congo Red has shown to be a possible carcinogen with the Ames Test, so proper protective equipment needs to be used. 

Gram Staining (1884)

Gram staining is often the first staining technique that microbiologists will learn. The purpose of this test is to identify bacteria belonging to one of two groups: gram-negative and gram-positive bacteria. Before molecular methods to identify a type of bacteria, gram staining was the first step in characterizing a microbe. 

Gram staining uses several dyes as well as an alcohol rinse. The process involves heat fixing the bacteria to a slide, submerging the slide in crystal violet, and followed with submerging in Lugol’s iodine. The iodine causes crystals to form, which remain trapped in the bacteria with the thick cell walls (hence gram-positive). In gram-negative bacteria, the crystal violet washes out with an alcohol rinse. In order to stain gram-negative bacteria, a counterstain such as fuchsin is used. It is important to note that dead or permeabilized gram-positive bacteria will allow crystal violet to wash out and will, therefore, appear to be gram-negative.

Trichrome Stain (1888)

Just as the name implies, this method uses three (tri) colors (chrome). There are several variations on this technique for specific techniques but in modern histology trichrome stain generally refers to Masson’s trichrome stain. Masson’s trichrome stain was developed to differentiate cells from the surrounding connective tissue, which is less pronounced in samples stained with H&E. Trichrome stain uses Fast Green FCF to stain the connective tissue. This method is particularly useful in muscular dystrophy research and other muscular diseases.

Giemsa Stain (1891)

Most commonly Giemsa stain is used for observation of blood smears for clinical analysis of blood disorders or infection. Giemsa staining is also useful for pathogen studies, which has been used to study malaria, Chlamydia, and cytomegalovirus to name a few. The Giemsa stain contains three chemicals: methylene blue, eosin, and Azure B. When combined with May Grunwald, different cells stain anywhere from dark blue to pale pink and even grey.

Giemsa stain binds AT-rich regions of DNA, but it doesn’t require a fluorescent microscope like DAPI. Chromosomal banding or “G banding” takes advantage of the stain’s preferential staining of AT regions creating a recognizable pattern on chromosomes. Chromosomal translocations found via Giemsa staining can be followed up with more precise techniques such as DNA sequencing or FISH.

Oil O (1926)

Oil O staining is comparatively the new kid on the block for histology stains. Oil O stain is in the family of “Sudan Stains” and binds triglycerides and lipids. This is used instead of the fluorescent compound Nile Red. Adipose tissue and fecal smears will use Oil O stain to get an estimate of lipid content, but the stain is not quantitative. Most protocols are developed for fresh or frozen tissues because lipid droplets are preserved best. If fixation is required, PFA is the best option for preserving lipid droplets because organic solvents strip cells of lipids.  

Less obvious uses for Oil O staining are lipid metabolism disorders (diabetes, atherosclerosis), viral infections, hormone biogenesis. 

Concluding Remarks

Good sample preparation and microscopy doesn’t have to be expensive. These dyes and many more will label structures and cost significantly less than immunofluorescence. Try them out for yourself!

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