connective tissue Flashcards

Question

big dye molecules

Answer 1

aniline blue light green acid fuchsin - too big to be absorbed into small pores

Answer 2

- different proteins form a mesh of variable pore size - RBCs ( small) - muscle/ cytoplasm ( medium ) - Collagen/ mucin ( large ) smaller dye can enter but will be pushed out and replaced by bigger one pH - 1.5- 3 proteins are basic at this pH( require low pH to stain connecive tissue fibers) Heat - increases the staining rate & influences penetration of large dye particles Pore size - or accessibility of reactive groups with dye

Answer 3

iron mordanted resistant to low pH - Weigerts - Verhoeffs - Celestine blue acidic component = iron mordant will be in combo with other stains in trichrome

Answer 4

NBF id NOT optimal but better staining can be achieved by post mordanting in bouins solution ( picric acid ) or mercuric chloride or both the longer a tissue is fixed in formalin the more staining is suppressed bc fixative takes up binding sites so less are available for stain

Answer 5

1.Small anionic dye is able to pass through all the pore sizes in the tissue - Everything is small dye color ( yellow) 2.Heterpolyacid fits through collagen pores and replaces the small anionic dye. - Cytoplasm remains the same, collagen and mucus appear unstained ( red) 3.Large molecule dye bonds with the heterpolyacid and stains collagen - Cytoplasm red, collagen and mucus blue (blue/green)

Answer 6

- most popular trichrome stain - used to differentiate collagen & smooth muscle tumors - identifies increases in collahem in diseases like cirrhosis - stains deposits of fibrin ( seen at site of injury )

Answer 7

No control is needed Tissues fixed long term in NBF will have poor staining ( occupies lots of binding sites ) Bouin’s is preferred ( contains picric acid)

Answer 8

Following hydration, mordant in Bouin’s 1 hour at 56°C Wash yellow colour out Stain in Weigert’s working solution Wash, then stain in small molecule dye – biebrich scarlet- acid fuchsin All tissue elements are now RED Rinse & apply heteropolyacid Binding with collagen occurs Red dye removed from collagen (appears unstained) Stain in larger molecule dye- aniline blue or light green Collagen is stained blue or green DCM

Answer 9

nuclei - black ( iron mordanted hematoxylin) cytoplasm, keratin, muscle, new fibrin - red ( small molecule dye) Collagen, mucus & old fibrin deposits- blue ( analine blue) or green ( light green)

Answer 10

Masson trichrome stain will be crowded with blue staining collagen collagen takes over in liver cirrhosis more fat than usual may also be seen

Answer 11

trichrome stain Hydrate Mordant in Bouin’s Stain with Weigert’s ( iron mordanted ) Stain with trichrome stain which contains (small molecule dye), PTA (heteropolyacid) and aniline blue or light green (large molecule dye)

Answer 12

nuclei- black cytoplasm, keratin, muscle- red Collagen & mucus - Blue not best choice but most used for Muscle Biopsies *

Answer 13

trichrome stain Stain contains acid fuchsin and picric acid Usually used as a counterstain **** results: Cytoplasm – Yellow Collagen- Red Nuclei – usually black due to iron hematoxylin

Answer 14

Trichrome stain Reliable FIBRIN staining ** Small dye- Martius Yellow Nuclei – Celestine blue or iron hematoxylin Medium dye – Crystal ponceau Phosphotungstic acid Large dye - aniline blue

Answer 15

Nuclei – Blue or Black Muscle – Paler Red Collagen – Blue Fibrin – Red  RBCs – Yellow

Answer 16

Used to demonstrate pathological changes in elastic fibers Thinning or loss – arteriosclerotic Breaks or splitting – other vascular disease Used for ID or examination of tumour invasion

Answer 17

Elastic fibers are stained but NOT specifically H&E HPS (hematoxylin phloxine saffron) Congo Red PAS

Answer 18

Specific or more intense stains include: Verhoeff’s ( most widely used) Aldehyde-fuchsin ( we use in lab) Resorsin Fuchsin  Orcein (oldest)

Answer 19

Eosin, Phloxine, or Congo Red stain through ***columbic reaction between elastin protein and acid dyes PAS stains ***glycoproteins in the fibers

Answer 20

Elastin fibers are ***cross-linked by disulfide bridges Oxidation occurs by ***permanganate in Aldehyde Fuchsin & Weigert’s Resorcin Oxidation occurs by ***iodine in Verhoeff’s Oxidation in both stains produces sulfonic acid derivatives to be formed. These sulfonic derivatives are strongly basophilic and this may account for staining of cytoplasm and other basic tissue components Reactions are enhanced by high electrolyte concentrations in stain solutions which inhibit uptake by chromatin, RNA etc. ( suppresses background staining)

Answer 21

specific stain but not top choice Some of the iron in the staining solution oxidizes the Hematoxylin, some oxidizes the elastin Verhoeff’s imparts black color to elastin, nuclei, also myelinated nerve fibers

Answer 22

Lugol Iodine (iodine, potassium iodide, H2O) - iodine is mordant & oxidizers ) 10 % ferric chloride - mordant differentiator; Regressive Stain** Alcoholic hematoxylin

Answer 23

Ferric chloride is used to remove colour until the elastic fibers are distinct (mordant differentiation) Sodium thiosulfate is used to remove the iodine Counterstain in van Gieson or other

Answer 24

elastic fiber stain Aldehyde fuchsin is made from alcoholic pararosaniline( AKA Basic fuchsin) , HCL & paraldehyde (or acetaldehyde) Deparaffinize to 70% alcohol ( NOT TO H2O bc stain is alcoholic) Stain, check microscopically. Some methods are first oxidized with ****permanganate & bleached with ***oxalic acid If not stained intensely enough return to stain, if too much, decolorize with alcohol Counterstain with light Green or other

Answer 25

A combination of staining techniques are employed in one method Stains mucin, fibrin, elastic fibers, muscle and collagen

Answer 26

elastic fiber stain Acid mucins are stained first Hydrate Stain in Alcian Blue Place slides in alkaline alcohol Converts alcian to monastral fast blue: insoluble Stain nuclei and elastin Stain in iron Hematoxylin (Verhoeff) Differentiate with ferric chloride Remove iodine with sodium thiosulfate Stain muscle, fibrin and collagen Stain in crocien scarlet- acid fuchsin - Small molecule dye Phosphotungstic acid - Removes red dye from collagen, fibrin, fibrinoid Stain with safran (saffron) - Stains collagen yellow

Answer 27

Nuclei & Elastin................Black Collagen.........................Yellow Ground substance & mucin....Blue Fibrinoid, fibrin.........Intense Red Muscle...............................Red

Answer 28

No dyes are employed Metallic ions are deposited on fibers and cells as an alternate method of providing contrast Solutions such as **ammoniacal silver are reduced by certain tissue elements

Answer 29

Silver *** usually silver Gold Osmium Mercury chromate( toxic) Palladium Lead Copper

Answer 30

Reticular fibers Bacteria (spirochetes) Fungi Neuropathology** Detection of: - Aldehydes - Calcium - Metal

Answer 31

chemically clean glassware! - can reduce silver = false positive solutions shouldn't contact metal surfaces - use gloved hand not forceps silver nitrate blackens skin & clothing silver solutions must be neutralized before discard ( becomes explosive over time if not neutralized) - NaCL can be used ( becomes cloudy) - HCl can be used (dilute) sections float off due to alkalinity of silver solution - histogrip or chemical waterbath prevents this store in a dark bottle in fridge make solutions in fume hood

Answer 32

Oxidizer – some methods require pre-oxidation Example: ****potassium permanganate produces aldehydes ( tuens section purple- follow with bleach) Sensitizer – empirical enhancement of silver deposits ( sometimes used) Bleach- Example: **oxalic acid following permanganate to remove purple colour

Answer 33

Stock solution- stable solution Working solution- must be made fresh using stock solution Reducer –reduces residual silver deposited at reaction sites Example: formaldehyde or Hydroquinone ( extraneous reducer)

Answer 34

Toner – silver may not provide enough contrast, gold is added using gold chloride Fixer – solution which removes unreduced silver. Sodium thiosulfate (hypo) these two are always used

Answer 35

ARGYROPHIL- metallic impregnation reactions which involve the use of an extraneous reducer -( Formaldehyde or hydroquininone) ARGENTAFFIN – metallic impregnation which needs no extraneous reducer - tissue is able to turn silver black Tissue components have chemical characteristics which make them either argyrophil or argentaffin. The reticulin staining method, Gordon and Sweet’s & Gomori’s are an argyrophil techniques. The extraneous reducer applied is Formaldehyde. Argyrophil methods will stain BOTH types of tissue elements

Answer 36

Hydrate to distilled (test and control) Oxidize in potassium permanganate Rinse Bleach (oxalic acid) OR remove excess with potassium metabisulfite Sensitize in ferric ammonium sulfate Wash...wash... wash Apply silver solution Quick rinse* Reduce with formaldehyde Wash Tone in Gold chloride (increase contrast & permanence) Rinse Apply potassium metabisulfite (Gomori’s) Fix in sodium thiosulfate Wash Counterstain if desired DCM

Answer 37

This is usually the make or break part of silver impregnation Involves silver nitrate and sodium or potassium hydroxide with ammonia Preparation takes patience

Answer 38

Used to demonstrate muscle cross striations, fibrin or glial fibers Hematoxylin with a tungsten mordant Zenker fixative is preferred If Zenker is used what are the implications?

Answer 39

Oxidize in permanganate Bleach in oxalic acid Stain overnight Dehydrate rapidly

Answer 40

PAS-Methenamine Silver Review method in book, note the use of microwave Jones Methenamine Silver is more user riendly

Answer 41

Similar to Grocott methenamine silver GMS Hexamine and methenamine refer to the same substance HEXAMETHYLENETETRAMINE This substance replaces the ammonia & other alkali

Answer 42

Selective solubility stains The dyes used are more soluble in lipid than the solvent they are in Paraffin sections cannot be used Alcohol & clearing agents remove lipids Solvent is important Isopropanol &/or propylene glycol Dye must be strongly colored

Answer 43

Oil Red O and Sudan black B are commonly used (not water soluble) Control not required Mashing the coverslip may remove dye from tissues

Answer 44

Fat emboli (following bone fracture or crushing of fatty tissue) Degenerating tissues Identification of liposarcoma Determine fat presence (stool, breast milk)

Answer 45

Osmium the fixative, preserves fat and makes it turn black If you see a paraffin section stained for fat this is the method Formalin fixed tissue may be washed, then placed in Osmium May be stained with any method ( fat is black before the staining) Gross amounts of fat will not be fixed Osmium has very limited penetration, face the block carefully

Answer 46

Toluidine Blue Methylene Blue stain - Metachromatic staining can be used for the demonstration of mucins, cartilage, mast cells, etc.. Methyl Green- Pyronin Y for plasma cells

connective tissue Flashcards

(70 cards)