STAINING
The process of applying dyes on the sections to study 1. Methyl violet or crystal violet
architectural pattern of the tissue and physical 2. Cresyl blue (for reticulocytes)
characteristics of the cells 3. Safranin
Different tissues and cells have varying affinities for 4. Bismarck brown
most dyes and stains 5. Basic fuchsin
AFFINITY: 6. Methylene blue
Acidic (nucleus) basic stains 7. Thionine
Basic (cytoplasm) acidic stains 8. Toluidine blue
9. Azure A, B, C
PROGRESSIVE STAINING
Tissue elements are stained in definite sequence Water is necessary for most metachromatic staining
The staining with specific periods of time or until techniques
desired color is attained Metachromasia is usually lost if section is dehydrated
Not washed or decolorized in alcohol after staining
The distinction of tissue detail relies solely on the Metachromasia is satisfactorily seen in formalin-fixed
selective affinity of the dye for various cellular tissues
elements
COUNTERSTAINING
REGRESSIVE STAINING Application of a different color or stain to provide
First over-stain the tissues to obliterate cellular details contrast and background to the staining of the
Excess stain is removed or decolorized from structural components to be demonstrated
unwanted parts of the tissue and until the desired
color is obtained Cytoplasmic stains
Red Yellow Green
DIFFERENTIATION/DECOLORIZATION Eosin Y Picric acid
Lt. Green SF
The selective removal of excess stain from the tissue Eosin B Orange G
Lissamine Green
during regressive staining so that a specific substance Phloxine B Rose Bengal
may stain distinctly from the surrounding tissue
Usually done by washing the section in simple Nuclear stains
solution (e.g. water or alcohol) or use of acids and Red Blue
oxidizing agents Neutral Red
Methylene blue
Primary stain = basic dye Safranin O
Toluidine blue
Differentiation = acidic solution Carmine
Celestine blue
Primary stain = acidic dye Hematoxylin
Differentiation = basic solution
1. Alcohol METALLIC IMPREGNATION
― Differentiator for both acidic and basic dyes The process where specific tissue elements are
by dissolving excess dye demonstrated not by stains but by colorless solutions
2. Mordant (e.g. iron alum) of metallic salts which are deposited on the surface of
― A differentiating agent the tissue
― Can oxidize hematoxylin to a soluble, It is not absorbed by the tissues, could be a
colorless compound precipitate or a reduction product on certain tissues
Disadvantage: if a mordant stained section is E.g. gold chloride, silver nitrate
allowed to remain in a differentiating agent such as
1% or 2% alcohol, all of the dye will be removed VITAL STAINING
o Restaining faded slides The selective staining of living cell constituents
Demonstrates cytoplasmic structures
METACHROMATIC STAINING ― By engulfment of the dye particle
Makes use of specific dyes which differentiate ― By staining of pre-existing cellular
particular substances by staining it with a color that is components
different from that of the stain itself (metachromasia) Nucleus is resistant to vital stains
Usually employed in staining cartilage, connective Two Types
tissue, epithelial mucins, amyloid and mast cell 1. Intravital staining
granules By injecting the dye into any part of the animal
Metachromatic dyes (basic) – belongs to thizine and body
triphenylmethane groups E.g. lithium, carmine and India ink
2. Supravital staining MAJOR GROUPS OF TISSUE STAINING
Used immediately after removal of cells from the 1. HISTOLOGICAL STAINING
living body The process whereby the tissue constituents are
E.g. Neutral red (best), Janus green demonstrated in sections by direct interaction with a
(mitochondria), Trypan blue, Nile blue, Thionine dye or staining solution
and toluidine blue Active tissue components are colored
E.g. micro-anatomical stains, bacterial stains, specific
ROUTINE HEMATOXYLIN AND EOSIN (H&E) tissue stains (e.g. muscles, connective tissue and
Most common method utilized for microanatomical neurologic stains)
studies of tissues 2. HISTOCHEMICAL STAINING (HISTOCHEMISTRY)
Uses the regressive staining which consists of The process whereby various constituents of tissues
a. Overstating the nuclei are studied thru chemical reaction that permits
b. Removal of superfluous and excessive color of microscopic localization of specific tissue substances
the tissue constituent by acid differentiation E.g. Perl’s Prussian blue reaction for hemoglobin and
Periodic Acid Schiff staining for carbohydrates
Enzyme Histochemistry
H and E PROCEDURE Active reagent – substrate
1. Clear paraffin embedded sections in first xylene bath for Tissue – enzymes
3 minutes The final opacity or coloration is produced from
2. Transfer to second xylene batch for 2 to 3 minutes the substrate rather than the tissue
3. Immerse in first bath of absolute ethyl alcohol for 2 3. IMMUNOHISTOCHEMICAL STAINING
minutes A combination of immunologic and histochemical
4. Transfer to a bath of 95% ethyl alcohol for 1 to 2 techniques that allow phenotypic markers to be
minutes detected by antibodies (e.g. polyclonal, monoclonal,
5. Rinse in running water for a minute enzyme-labeled or fluorescent labeled)
6. Stain with Harris Alum Hematoxylin for 5 minutes
(Ehrlich’s hematoxylin requires 165-30 minutes)
7. Wash in running tap water to remove excess stain METHODS OF STAINING
8. Differentiate in 1% acid-alcohol (1mL conc. HCl to 99 1. Direct Staining
mL of 80% ethyl alcohol) for 10-30 seconds until the Uses aqueous or alcoholic dye solutions (e.g.
nuclei are stained methylene blue, eosin) to produce a color
9. Rinse in tap water 2. Indirect Staining
10. Use ammonia water (average of 5 minutes) or 1% Uses a mordant or another agent to intensify the
aqueous lithium carbonate until the section appears action of the dye used
blue (about 30 seconds) MORDANT
11. Wash in running water for 5 minutes ― Serves as a link or bridge between the tissue
12. Counterstain with 5% aqueous eosin for 5 minutes. If and the dye
alcoholic eosin is used, the time can be reduced to 30 ― The dye may stain weakly by itself, therefore
seconds or 1 minute the mordant combines with the dye forming a
13. If aqueous eosin is used, wash and differentiate in tap colored “lake” which would combine with the
water under microscopic control until the nuclei appear tissue forming an insoluble “tissue-mordant-
sharp blue to blue black and the rest is in shades of dye-complex”, which would allow subsequent
pink. If alcoholic solution is used, differentiate with 70% counterstaining and dehydration
alcohol ― E.g. Potassium alum with hematoxylin in
14. Dehydrate, clear and mount Ehrlich’s hematoxylin. Iron in Weigert’s
hematoxylin
H& E RESULT ACCENTUATOR
Nuclei – blue to blue black ― Not essential and does not participate to the
Karyosome – dark blue chemical reaction of the tissue and dye
Cytoplasm – pale pink ― Accelerates the speed of the staining reaction
RBCs, eosinophilic granules, keratin – bright-orange red by increasing the staining power and selectivity
Calcium and decalcified bone – purplish blue of the dye
Decalcified bone matrix, collagen and osteoid – pink ― E.g. potassium hydroxide in Loeffler’s
Muscle fiber – deep pink methylene blue, phenol in Carbon thionine and
Carbol fuchsin