Common Cellular Stains
An interactive page summarizing the common cellular staining techniques and stains that act on different cellular components, including plasma membrane, cell wall, cytosol, nucleus, connective tissues and various cell organelles.
Dyes used for staining have a greater affinity for tissue molecules than solvent molecules. Level of affinity depends on the structure, shape, and charge distribution of the dye and the solvent properties.
Acidic (pH < 7) stains provide H+ to tissue molecules and adds to their positive charge, while basic (pH > 7) stains removes the positive charge.
Mouse over (or click and observe below the illustration) the cell component to know about the normal stain (white background) and fluorescent stain (black background) with the colour observed.
Peroxisome Stains
Alexa Fluor 488
PMP-70 GFP
Diaminobenzidine (DAB)
Pyronin B
Lysosome Stains
Nucleolus Stains
NP-mPB-tGFP
(nucleolus predominant mammalian codon optimized PBase transposon GFP)
PARP-2
PARP-1
Azure B
Centrosome Stains
Pericentrin Antibody
Ninein Antibody
Microfilaments Stains
Fast Green FCF
Phalloidin, Phallacidin
Lysosome Stains
Nuclear Yellow
Acridine Orange
Redoxsensor Red CC1
DiSC3(5)
Ethyl Eosin
Cathepsin B probe
Neutral Red
Plasma Membrane Stains
FM 4-64
Cyanotolyl Tetrazolium Chloride (CTC)
Vacuole Stains
Bismark Brown Y
Dansyl Cadaverine (autophagy)
NBD C6-Ceramide
Lucifer Yellow CH
Cytoplasm Stains
Pyronin B
Basic Fuchsin
Eosin Y
Brilliant Green
Fast Green FCF
Light Green SF Yellowish
Microtubule Stains
DiOC6(3)
Golgi Apparatus Stains
Acridine Orange
Bodipy FL C5-Ceramide
NBD C6-Ceramide
Intermediate Filaments Stains
Cyanine-3
Cyanine-3.5
Cyanine-5
Cyanine-5.5
Cyanine-7
Cyanine-7.5
Rhodamine B
Vimentin Monoclonal Antibody (V9)
Cytokeratin Pan Monoclonal Antibody(C11)
Alexa Fluor
RER Stains
Bodipy FL C5-Ceramide (boron-dipyrromethene)
DiOC6(3)
Chromatin Stains
TOTO 1
YO-PRO 1
YOYO 1
BOBO 3
TO-PRO 3
Ethidium Monoazide
Toluidine Blue O
Alcian Blue 8 GX
Carmine
Secretory Vesicles Stains
Acridine Orange
FM 4-64
Lucifer Yellow CH
Nucleus Stains
Acridine Orange (cell cycle)
Ethidium Monoazide
Hoechst
DAPI
Giemsa
Hematoxylin
Toluidine Blue O
Eosin Y
Ethyl Green, Methylene Green
Mitochondria Stains
DiOC6(3)
DiOC7(3)
JC-1
JC-9
Lucigenin
Nonyl Acridine Orange
Nuclear Yellow
Rhodamine 123
Dihydrorhodamine 123
DASPEI
Newport Green PDX
Janus Green B
Safranin O
TMRE, TMRM
Oxonol V
Redoxsensor Red CC1
Rhodamine 6G
Dihydrorhodamine 6G
DiSC3(5)
Ethyl Eosin
Lucifer Yellow CH
DASPMI
Methyl Blue
Oil Red O
Plasma Membrane Stains
FM 4-64
Cyanotolyl Tetrazolium Chloride (CTC)
SER / Lipids / Phospholipids Stains
Nuclear Yellow
Acridine Orange
Redoxsensor Red CC1
Di-8-ANEPPS
DiI
DiSC3(5)
Ethyl Eosin
Bodipy FL C5-Ceramide
DiSC3(5)
FM 4-64
BOBO-1
DiSBAC2(3)
Lucifer Yellow CH, CV
DASPEI
NBD C6-Ceramide
Neutral Red
Oil Red O
Sudan III , Sudan IV
Orcein
Basic Fuchsin
Giemsa
Hematoxylin
Nile Blue A
Sudan Black B
Mitochondria Stains
Cytoplasm Stains
Eosin Y
SER / Lipids / Phospholipids Stains
Nuclear Yellow
Acridine Orange
Redoxsensor Red CC1
Di-8-ANEPPS
DiI
DiSC3(5)
Ethyl Eosin
Bodipy FL C5-Ceramide
DiSC3(5)
FM 4-64
BOBO-1
DiSBAC2(3)
Chromatin Stains
Nucleus Stains
Common Staining Techniques
Gram
Masson's Trichrome
Acid-Fast
H & E
Papanicolaou
Gram Staining
Gram-positive bacteria have a thick mesh-like cell wall made of peptidoglycan (50–90% of cell envelope), and as a result are stained purple by crystal violet, whereas gram-negative bacteria have a thinner layer (10% of cell envelope), so do not retain the purple stain and are counter-stained pink by safranin.
Pre-staining heat fixation affixes bacteria to the slide without compromising vitality in order to prevent rinsing during staining.
Step 1 : Crystal violet (CV) dissociates in aqueous solutions into CV+ and chloride (Cl−) ions. These ions penetrate the cell wall of both gram-positive and gram-negative cells. The CV+ ion interacts with negatively charged components of bacterial cells (teichoic acid and lipoteichoic acid) and stains the cells purple.
Step 2: Iodide(I− ) ion from Lugol's Iodine (a mixture of KI and iodine in water) interacts with CV+ and forms large complexes of crystal violet and iodine (CV–I) within the inner and outer layers of the cell. It traps CV-I complex and prevents its removal on addition of the decolorizer alcohol.
Step 3: When a decolorizer (alcohol or acetone) is added, it interacts with the lipids of the cell membrane.
A gram-negative cell loses its outer lipopolysaccharide membrane as Braun's lipoprotein (a covalently linked molecule to peptidoglycan that attached to the outer membrane) is hydrolyzed. The inner peptidoglycan layer is left exposed. The CV–I complexes are washed from the gram-negative cell along with the outer membrane.
A gram-positive cell becomes dehydrated from an ethanol treatment. The large CV–I complexes become trapped within cell due to the multilayered nature of its peptidoglycan.
Step 4: Positively charged counterstain (safranin or basic fuchsin), is applied to give decolorized gram-negative bacteria a pink or red color. Both gram-positive bacteria and gram-negative bacteria pick up the counterstain, which is less visible in gram-positive bacteria because of the retention of darker crystal violet stain.
Masson's Trichrome Stain
It follows one of the two principles in staining -
(1) less porous tissues are colored by the smallest dye molecule, and if a larger dye is able to penetrate, it will replace the smaller dye. OR
(2) less permeable components retain Biebrich scarlet which initially binds to acidophilic components, while it is pulled out of the more permeable collagen. After phospho acid treatment, collagen binds with aniline blue.
It is a combination of 4 solutions -
1. Weigert's iron hematoxylin - it is made up of 1 : 1 volume mixture of two solutions
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Solution A : 5 g hematoxylin + 500 mL 95% alcohol
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Solution B : 20 mL of 29% FeCl3 + 475 mL of distilled water + 5 mL of HCl
2. Plasma stain - it contains
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2.7 g Biebrich Scarlet + 0.3 g acid fuchsin + 300 mL distilled water + 3 mL glacial acetic acid
3. Phospho acid mixture -
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25 g phosphotungtic acid + 25 g phosphomolybdic acid + 1000 mL distilled water
4. Fibre stain -
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2.5 g aniline blue + 100 mL distilled water + 1 mL 1% glacial acetic acid
Stain is later fixed using Bouin's fixative,
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1500 mL saturated picric acid + 500.0 mL formaldehyde + 100.0 mL glacial acetic acid
Haematoxylin and Eosin (H & E) Stain
It is the most widely used staining in medical diagnosis. Haematoxylin (H), a cationic stain stains nuclei blue and eosin (E) stains extra-cellular matrix and cytoplasm pink.
H oxidizes to hematein in the presence of sodium iodate and binds to Al 3+ (found in mordant molecules) which makes it a positively charged complex. This complex binds to the negatively charged molecules like DNA backbone in chromatids.
Haematoxylins are classified based on the mordant (substance that fixes the dye on the tissue through coordinated complexes) and oxidation method used, as shown in table below.
Eosin Y is an acidic and synthetic xanthene dye derived from fluorescein. It binds to positively charged residues (arginine, lysine, histidine) in cytosol, nucleolus and connective tissues. Optimum staining pH is 4-5.
The basic protocol involves
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Deparaffinization - prior to creating slices of tissues to be observed, they are placed in paraffin wax and cooled. Thereafter, using precise sectioning tools in a microtome, thin tissue slices embedded in paraffin are obtained. This paraffin needs to be dewaxed before the tissue can be observed after staining. Slices are treated with Xylene two times for 2-3 minutes.
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Hydration - using graded alcohols from 100% to 95% to 70%, tissues are dehydrated to remove residual paraffin. Tissues are then washed with water to rehydrate them and remove residual alcohol.
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Staining
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Haematoxylin - hematein in this stain colors the nuclei blue.
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Differentiation - tissues are rinsed in a weak acid (0.5-1% HCl diluted with water or 70% ethanol) to remove excess staining. It breaks bonds between hematein, Al3+ and tissues. Usually, this is applied in regressive stains (Harris's), but also in progressive stains (Mayer's and Gill's), in which only a limited haematoxylin is applied to remove background and mucin staining and intensity is varied with time. Differentiation optimizes contrast.
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Bluing - blue color is established in tissues by fixing it in an alkaline ammonia water buffer (pH 8.0-8.2), lithium carbonate or Scott's tap water (magnesium sulphate buffered with sodium bicarbonate). It converts coloration from reddish-purple to blue. Increased pH removes H+ from hematoxylin ring that changes its color.
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Eosin - tissue is counterstained with eosin Y. Eosin renders pink and red colors to the tissues. An alcohol rinse is required in case one uses alcoholic eosin.
4. Dehydration - tissues are dehydrated using graded alcohol from 70% to 95% to 100%.
5. Clearing - tissues are rinsed with xylene to convert the medium non-aqueous from alcoholic.
6. Mounting - An organic mounting medium (e.g. limonene) does not affect color nor tissue features, and has excellent optic and preservative properties. After mounting and drying (xylene is evaporated), sections can be observed at light microscopy.
Shown below is the hypothesized mechanism of Masson's trichrome stain.
Acid Fast (Ziehl–Neelsen) Staining
Mycobacteria have a thick cell wall consisting of highly hydrophobic lipid components - mycolic acid and arabinogalactan, the latter being a highly strong and stiff biopolymer and is a part of plant gums. Thus, these mycobacteria are not affected by the application of decolorizing agents like alcohol. In contrast, other Gram negative bacteria are de-stained after applying the decolorizer.
Staining procedure is as follows -
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Stain the smear with carbol fuchsin. It solubilizes the lipid material present in the Mycobacterial cell wall.
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By the application of heat, it further penetrates through lipid wall and enters into cytoplasm. Then after all cell appears red.
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The smear is decolorized with decolorizing agent (3% HCl in 95% alcohol) but the acid fast cells are resistant due to the presence of large amount of lipid material in their cell wall which prevents the penetration of decolorizing solution. The non-acid fast organism lack the lipoidal material in their cell wall due to which they are easily decolorized, leaving the cells colorless.
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The smear is stained with counterstain, methylene blue. Only decolorized cells absorb the counter stain and take its color and appear blue while acid-fast cells retain the red color.
Papanicolaou Staining (Pap Stain)
Pap stain distinguishes between basophilic and acidophilic cell components and obtains a detailed chromatin pattern. It is used in oral and cervical cancer screening.
The pap stain has 3 solutions having 6 dyes -
Solution 1 : Harris's Hematoxylin - basic nuclear stain.
Solution 2: OG-6 (Orange gelb 6) - acidic cytoplasmic stain that stains keratin, where 6 denotes the concentration of phosphotungstic acid (mordant).
Solution 3 : Eosin Azure 36/50 - polychromatic stain with pH 4.5-5 consisting of
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Light green SF - acidic cytoplasmic dye that stains metabolically active cells green
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Intermediate squamous cells
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Parabasal cells
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Endocervical cells
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Histiocytes
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Leukocytes
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Undifferentiated carcinoma cells
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Adenocarcinoma cells
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Eosin Y - acidic cytoplasmic dye that stains red
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Superficial squamous cells
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Nucleoli
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Erythrocytes
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Cilia
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Bismarck brown Y - precipitates phosphotungstic acid
It is classified into two staining methods -
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Progressive Pap staining - nucleus is stained to the intensity required followed by bluing agent. It uses Mayer's or Gill's hematoxylin.
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Regressive Pap staining - nucleus is over stained using Harris's hematoxylin, the excess stain is removed by dilute HCl. It is more common and stable method, as shown below.