Adhesive, Staining, and Labelling

What is adhesion?

Is the process of ensuring that the tissue sections remains firmly attached to the glass slide during staining and subsequent handling.

Tissue maybe attached to slides by: methods of achieving adhesion:

1. Incubator drying 2. Wax oven drying 3. Hot plate drying 4. Use of chemical adhesive

Incubator drying temp

37C overnight

Wax oven drying temp:

56-50-C about 2 hours

Hot plate drying

45-55C (30-45 minutes)

Easy to prepare, Inexpensive, widely used

Mayer’s egg albumin

Convenient alternative, maybe added to the flotation bath

Gelatin 1percent

Useful coated slide adhesive

Gelatin-formaldehyde mixture

Commonly used in immunohistochemistry, strong adhesiuve property, coated slides should be used within a few days

Poly-L-Lysine

Stronger and long lasting adhesion, especially useful in cytology and bloody/proteinacous specimens.

APES (3-Amino-Propyl-Tri-Ethoxy-Silane)

3 major groups of staining

1. Histological staining

2. Histochemical staining

3. Immunohistochemical staining

Tissue constituent are stained by direct interaction with a dye or solution

Histological staining

Chemical reactions of a specific tissue substance with a dye

Histochemical staining

• Combinations of immunologic and histologic staining

• Phenotypic markers were detected and seen through the microscope

Immunohistochemical staining

Types of staining

1. Direct staining

2. indirect staining

3. Progressive staining

4. Regressive staining

5. Metachromatic staining

6. Counterstaining

7. Metallic impregnation

8. Vital staining

9. ntravital staining

10. Supravital staining

11. Hematoxylin and Eosin staining.

H&E STAINING: NUCLEI:_____

BLUE TO BLUE BLACK

H&E STAINING: KARYOSOME:_____

DARK BLUE

H&E STAINING: CYTOPLASM:_____

PALE PINK

H&E STAINING: RBC, EOSINOPHILIC GRANULES & PROTEIN:_____

BRIGHT ORANGE - RED

H&E STAINING: CALCIUM AND DECALCIFIED BONE:_____

PURPLISH BLUE

H&E STAINING: DECALCIFIED BONE MATRIX, COLLAGEN, AND OSTEOID:_____

PINK

H&E STAINING: MUSCLE FIBERS:_____

DEEP PINK

OTHER STAINING TECHNIQUES:

1. Heidenhains Iron Hematoxylin method

2. Celestine Blue-Haem alum sequence staining

3. Mallory’s phloxine methylene blue staining

cellnuclei, cytoplasmic inclusions and muscle striations in Heidenhain’s iron hematoxylin method will appear what color:

BLACK

An oxazine dye used as an alternatieve to Iron Hematoxylin nuclear stain

Celestine blue-haem alum sequence staining

It forms a lake with iron alum

Celestine blue

Acts as a mordand to bind hematoxylin

Celestine blue

Celestine blue forms _____ with____to bind _____

LAKE WITH IRON ALUM TO BIND HEMATOXYLIN

Originally known as Eosin-methylene blue method

Mallory’s Phloxine Methylene blue staining

Histopathologic dyes: 2 types

1. Natural dyes

2. Artificial dyes

Natural dyes:

1. Hematoxylin

2. Cochineal dyes

3. Orcein

Artificial dyes

1. Acid dyes 2. Basic dyes 3. Neutral dyes 4. Hematoxylin types 5. Phosphotungstic acid hematoxylin 6. Lead hematoxylin 7. Eosin

EOSIN FORMS:

1. EOSIN Y

2. EOSIN B

3. EOSIN S

4. 5% AQUEOUS E EOSIN Y

5. ALCOHOLIC EOSIN

Most commonly used.

• Soluble in water, less soluble in alcohol.

• Available in Aqueous and Alcoholic solutions

  • *Green yellow flourescence especially in alcoholic medium

EOSIN Y

A.K.A. Erythrosin B; Deeper red color

EOSIN B

AKA Ethyl Eosin; Alcohol Soluble

EOSIN S

Eosin Y dissolved in water with thymol crystal to avoid fungal growth

5% Aqueous E eosin Y

Eosin Y dissolved in water with _____ to avoid fungal growth

THYMOL CRYSTAL

• Eosin Y dissolved in water

• Add Alcohol

• Add Glacial Acetic Acid

  • Combining EOSIN Y AND PHLOXINE B - PRODUCES A CYTOPLASMIC STAIN

ALCOHOLIC EOSIN

COMBINING EOSIN Y AND _____ =

PHLOXINE B = CYTOPLASMIC STAIN

• Picric Acid

• Van geison’s stains; demonstrate connective tissue

Acid Fuchshin

• Basic acridine flourochrome:

  • Differentiates dead and living cells

    • DNA shows Green Flourescence

    • RNA shows Red Flourescence

Acridine Orange

deposits of calcium salts and possible site of phosphatase activity

Acridine red 3B

• Complex, water-soluble phthalocyanine dye (similar to chlorophyll)

  • Staining mucopolysaccharides; more specific for connective tissues and epithelial mucin due to its use as an acid solution

Alician Blue

Contrast stain for Gram’s technique, Acid fast and papanicolau method for diptheria

Bismarck Brown

• Chromatin stain for fresh smears

• Slightly soluble in water of at neutral reaction

• Kept in ammoniacal solution

  • Combined with aluminum chloride to stain glycogen

Carmine

• Best known a an indicator

• Stain for axis cylinders in embryos

• Used as 4% aqueous solution in Krajians method (elastic tissues, amyloid and myelin)

Congo red

• Nuclear or chromatin stain

• Used in amyloid in frozen sections and platelets in blood

  • Gentian violet - Mixture of crystal violet, methyl violet, Dexterin

Crystal Violet

Staining blood smears, differentiates leukocytes

Giemsa stain

Metalic impregnation; made up of gold chloride and mercuric chloride

Gold impregnation

• Oldest stain; originally for starch granules

• stains amyloid, cellulose, starch, carotenes, and glycogen.

• Removal of mercuric fixatives artifact pigments

• Mordant use in gram’s staining

  • Grams iodine - Iodine with potassium iodide, distilled water; used in Gram Weigert method (for microbes, and fibrin in tissue sections)

    • Lugol’s Iodine - Test for Glycogen, amyloid, and corpora amylacea

IODINE

For microbes and fibrin in tissue sections

Gram’s iodine

Test for glycogen, amyloid, and corpora amylacea

Lugol’s Iodine

For mitochondria during intravital staining

Janus Green

Stain: ascaris eggs and RBCs; bacterial spore stain.

• Decolorizer

• and Counterstain

Malachite Green

Stains: Chromatin green in presence of an acid

Reaction with MUCIN: False Positive

Methyl green

Basic nuclear stain. Best employed with EOSIN.

• POLYCHROMING - oxidation of methylene blue. Results to the loss of methyl groups and leaving lower homologues of the dye (AZURES) and deaminized oxidation of products (Thiazoles).

• Lofflers Polychrome methylene blue - for rapid diagnosis and frozen section

Methylene blue

Oxidation of methylene blue. Results to the loss of methyl groups and leaving lower homologues of the dye (AZURES) and deaminized oxidation of products (Thiazoles).

POLYCHROMING

for rapid diagnosis and frozen section

Lofflers Polychrome methylene blue

Excellent stain for elastic fibers (Taenzer Unna Orcein Method)

For dermatological studies (demonstrates finest and delicate fibers in the skin)

Orcein

Staining fat; fats reduce osmium tetroxide to osmium dioxide (black staining of fat)

Osmium Tetroxide

Employed as a contrast stain to acid fuchsin. Demonstrates connective tissues

Picric acid

Colored salt of ferric ferrocyanide, normally utilized for manufacturing paints.

Intravital staining for circulatory system

Prussian Blue

Used with osmic acid. stains blood and glandular tissue

Rhodamine B

10% aqueous solution; for spirochetes, reticulum and fiber stains

Silver Nitrate

Nuclear stains for fixed tissues. Substitute for thionine in fresh frozen tissue sections.

Staining for nissl granules or chromophilic bodies.

Toluidine blue

Demonstrate neuroglia in frozen sections

Victoria Blue

Not a real dye because they do not have the auxochrome groups. Give colors to lipids because they are more soluble to lipid medium of the tissue.

Oil soluble dyes (LYSOCHROMES)

Most sensitive of all oil soluble dyes. Has more greater affinity for phospholipids than other lysochromes

Colors neutral lipids like Triglycerides

• Staining ability relies on the dye’s concentration, temperature, and physical state of the fats.

  • Sudan B - stains phospholipids and neutral fats doesn’t stain crystalline cholesterol and free fatty acids tends to be soluble in the ethanolic dye bath.

Sudan Black

Or Scharlach R. Has no secondary amino group. Doesn’t color phospholipids or the fine lipids droplets.

• Add benzoic acid - intensifies fat and prevent rapid deterioration of the solution. Recommended for staining triglycerides - giving them deep and intense red color.

SUDAN IV

First sudan dye introduced into histochemistry. Fat soluble, good as a fat stain for central nervous system tissue

SUDAN III