Common Cellular Stains

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

Microfilaments Stains

Fast Green FCF

Phalloidin, Phallacidin

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

Plasma Membrane Stains

FM 4-64

Cyanotolyl Tetrazolium Chloride (CTC)

Microtubule Stains

Mitochondria Stains

Nonyl Acridine Orange

Golgi Apparatus Stains

Acridine Orange

Bodipy FL C5-Ceramide

NBD C6-Ceramide

Centrosome Stains

Pericentrin Antibody

Ninein Antibody

RER Stains

Bodipy FL C5-Ceramide (boron-dipyrromethene)

Cytoplasm Stains

Eosin Y

Light Green SF Yellowish

Peroxisome Stains

Alexa Fluor 488

Diaminobenzidine (DAB)

Pyronin B

Intermediate Filaments Stains

Vimentin Monoclonal Antibody (V9)

Cytokeratin Pan Monoclonal Antibody(C11)

Nucleolus Stains

Secretory Vesicles

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

Vacuole Stains

Dansyl Cadaverine (autophagy)

Chromatin Stains

Nucleus Stains

Acridine Orange (cell cycle)

Ethyl Green, Methylene Green

Common Staining Techniques

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

Solution A : 5 g hematoxylin + 500 mL 95% alcohol
Solution B : 20 mL of 29% FeCl3 + 475 mL of distilled water + 5 mL of HCl

2. Plasma stain - it contains

2.7 g Biebrich Scarlet + 0.3 g acid fuchsin + 300 mL distilled water + 3 mL glacial acetic acid

3. Phospho acid mixture -

25 g phosphotungtic acid + 25 g phosphomolybdic acid + 1000 mL distilled water

4. Fibre stain -

2.5 g aniline blue + 100 mL distilled water + 1 mL 1% glacial acetic acid

Stain is later fixed using Bouin's fixative,

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

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.
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.
Staining

Haematoxylin - hematein in this stain colors the nuclei blue.
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.
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.
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 -

Stain the smear with carbol fuchsin. It solubilizes the lipid material present in the Mycobacterial cell wall.
By the application of heat, it further penetrates through lipid wall and enters into cytoplasm. Then after all cell appears red.
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.
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

Light green SF - acidic cytoplasmic dye that stains metabolically active cells green
- Intermediate squamous cells
- Parabasal cells
- Endocervical cells
- Histiocytes
- Leukocytes
- Undifferentiated carcinoma cells
- Adenocarcinoma cells
Eosin Y - acidic cytoplasmic dye that stains red
- Superficial squamous cells
- Nucleoli
- Erythrocytes
- Cilia
Bismarck brown Y - precipitates phosphotungstic acid

It is classified into two staining methods -

Progressive Pap staining - nucleus is stained to the intensity required followed by bluing agent. It uses Mayer's or Gill's hematoxylin.
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.

Common Staining Techniques

Gram

Masson's Trichrome

Acid-Fast

H & E

Papanicolaou

Gram Staining

​

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

Solution A : 5 g hematoxylin + 500 mL 95% alcohol​

Solution B : 20 mL of 29% FeCl3 + 475 mL of distilled water + 5 mL of HCl ​​

2. Plasma stain - it contains​​

2.7 g Biebrich Scarlet + 0.3 g acid fuchsin + 300 mL distilled water + 3 mL glacial acetic acid

3. Phospho acid mixture - ​

25 g phosphotungtic acid + 25 g phosphomolybdic acid + 1000 mL distilled water

4. Fibre stain - ​

2.5 g aniline blue + 100 mL distilled water + 1 mL 1% glacial acetic acid

​

Stain is later fixed using Bouin's fixative,

1500 mL saturated picric acid + 500.0 mL formaldehyde + 100.0 mL glacial acetic acid

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.

1. Weigert's iron hematoxylin - it is made up of 1 : 1 volume mixture of two solutions

Solution A : 5 g hematoxylin + 500 mL 95% alcohol

Solution B : 20 mL of 29% FeCl3 + 475 mL of distilled water + 5 mL of HCl

2. Plasma stain - it contains

3. Phospho acid mixture -

4. Fibre stain -