Histotechnology - Connective Tissue, Carbohydrates, Lipids

Purpose of 4 important reagents in PAS stain?

1. Periodic acid: mild oxidizer, turns carboxyl groups into aldehydes

2. Schiff reagent: HCL & sodium metabisulfate

3. HCL: Loss of chromophore

4. Running Tap Water: removes HCl and unbound schiff, allows formation of new chromophore

Purpose of 2 important reagents in Alcian Blue 2.5 stain?

1. 3% acetic acid: Protects stain from pH changes in distilled water

2. Alcian blue: Reacts with acid mucopolysaccharides (carboxylated and sulphated)

Purpose of important reagent in Alcian Blue 1?

0.1N HCL: ionize different groups on the section – so that when alcian blue is applied only the sulphated acid mucos will stain

Purpose of important reagent in Mucicarmine stain?

Mucicarmine: contains red dye bound to aluminium Aluminium forms chelation complex with dye making it pos charged so its attracted to negatively charged residues.

Purpose of 4 important reagents in Colloidal Iron stain?

1. 12% acetic acid: prevent dilution of colloidal iron solution

2. 2. Colloidal iron: ferric ions are positive so they are attracted to neg charged residues

3. Perls/ Ferro-cyanide: Reacts with the iron to produce colour

4. Nuclear fast red: counterstain

Purpose of 3 important reagents in Congo red stain?

1. Weigerts/ harris hematox: stain nuclei blue

2. Alkaline salt solution: align hydrogren bonding sites, release internal hydrogen bonds to increase reaction sites

3. Congo red: stains the amyloid red

Purpose of important reagent in Crystal violet for amyloid?

Crystal violet: polychromatically reacts with something in the amyloid and it stains it purple

Purpose of 2 important reagents in Thioflavin T stain?

1. Mayers hematoxylin: quench background nuclear fluorescence

2. Thioflavin T: attaches to amyloid, with yellow/green fluorescence

Purpose of 5 important reagents in Masson trichrome?

1. Bouins/Zenker: sections should be mordanted in either one to increase basophilia to increase staining intensity

2. Weigerts: progressively stain nuclei

3. PMA/PTA: differentiate- displace red from collagen

4. Light green/aniline blue: counterstain

5. Acetic acid: remove unbound dye molecules

Purpose of 4 important reagents in Verhoeff stain?

1. Verhoeffs iron hematox: 5% alc hematox, 10% ferric chloride, verhoeffs iodine-

2. Ferric chloride: differentiator and mordant

3. 5% Hypo: decolorizer, remove excess iodine stain

4. Van Gieson: counterstain

Purpose of important reagent in Toluidine blue stain?

Toluidine blue: metachromatic dye that stains chromotropes red/purple

Purpose of 5 important reagents in Gomori aldehyde fuchsin stain?

1. Aldehyde fuchsin: positively charged, stains elastic fibers Contains

2. paraldehyde (mordant)

3. Lugols iodine: oxidize elastic tissue

4. 5% Hypo: decolorizer, remove excess iodine

5. Light green/ van Gieson: counterstain

Purpose of 7 important reagents in Gordon & Sweets silver impreg stain?

1. Potassium permanganate: oxidizer, exposes aldehyde groups on reticulin

2. Oxalic acid: removes the color from potassium perming

3. Iron Alum: sensitizer, allows metallic ions to bind to neg charged aldehyde groups in reticulin

4. 10% formalin: reduce the silver ions (argyrophilic)

5. Gold chloride: for toning- tones down bg, get more refined texture

6. 5% hypo: fixes reduced silver in position, binds with unreduced silver to prevent non specific blackening

7. Nuclear fast red: counterstain

Purpose of 3 important reagents in Oil red O stain?

1. Isopropanol/ propylene glycol: introduce dye solvent into section, prepares it

2. Oil red O: dye and isopropanol, when applied to tissues it follows selective solubility- oil red O is more soluble in tissues than the isopropanol so itll go there Must be carried out in closed container or else it will precipitate

3. Ammonia water: blueing step

Purpose of 2 important reagents in Osmium tetroxide stain?

1. Osmium tetroxide- 5ml with 2mm tissue section placed in it for 1-2H. it will chemically combine with fat and blackening it

2. Periodic acid: differentiator- clears background of black stain where only the fats remain stained black

Purpose of 3 important reagents in Sudan black B stain?

1. Propylene glycol: helps dye bind to lipids without dissolving them Also acts as differentiator here to remove non specific bg staining and unbound dye

2. Sudan black B: to stain the fats via selective solubility

3. Nuclear fast red: counterstain