connective tissue Flashcards
what are the 4 basic tissue types
connective, epithelial, muscular & nervous
what is connective tissue
consists of cellular portion surrounded by non cellular ground substance
provides support & connects tissues
cells of connective tissue
fibroblasts
mast cells
histocytes
adipose tissue
reticular cells
osteoblasts & osteoclasts
chrondroblasts & chrondrocytes
blood cells
fibroblasts
secrete extracellular matrix components
- usually collagen & elastin
macrophages
tissue phagocytes
aka histocytes
tissue monocytes
reticular fibers
delicate fibers
framework fro more cellular organs ( Lymph nodes, spleen, liver)
indistinct in H&E :(
weakly birefringent
use special stains
- PAS(reticulin is carbohydrate rich )
- Argyrophil silver stain ( needs extraneous reducer) q
cartilage vs bone
cartilage lacunae are much larger
cartilage typically appears blue or light purple sue to neg charge (H&E)
fibrous structures (3)
collagen ( strength )
- muscles
elastin ( flexibility )
- skin/blood vessels
reticulin ( support mesh)
- around cellular organs ( Lymph nodes, spleen etc)
most abundant protein in the human body and where its found
collagen
stength to structures
found mostly in tendons, ligaments, skin ( fibrous tissues)
in smooth muscle, blood vessels, heart & gall bladder
collagen
very eosinophilic
birefringent
strength *
may be
- dense regular ( tendons, capsules & skin )
- loose irregular ( gallbladder)
basement membrane
basal lamina
barrier between epithelium & connective tissue
stained with carbohydrate techniques(PAS) or silver stains
JMS
Muscle ( 3 types)
skeletal
- nuclei pushed to periphery
- striated, involuntary
cardiac
- central nuclei & intercalated discs
- striated involuntary
smooth
- non striated, voluntary
elastin
key protein of extracellular matrix & main component of elastic fibers
highly flexible ( Balloon )
ex. blood vessels, skin
fibrin
most commonly seen after tissue damage
part of acute inflammation
involved in clotting
fibrinoid
acellular homogenous material
similar to fibrin ( but in different disorders)
found normally in placenta
formed in connective tissue or walls of blood vessels in some diseases
connective tissue stains are used to asses
Replacement of normal tissue with
connective tissue
Tumors
Basement membranes
Elastic or reticulin fiber
Fibrin or fibrinoid
Muscle
Connective tissue cells
types of connective tissue stains
Trichrome stains
Reticulin stains
Elastin stains
Basement membrane stains
Fat stains
Metachromatic stains for mast cells
Methyl green-pyronin Y
trichrome stains
differential staining
demostrate muscle & collagen differentiation
- possibly fibrin & erythrocytes
3 dyes as a minimum ( one must be a nuclear stain )
in trichrome stains when is an iron mordant used
when using Hematoxylin because trichrome stains have acidic components
Weigert -van Gieson Stain
Trichrome stain ( not a top choice )
- different results than usual
nuclei = black ( stained with iron hematoxylin )
cytoplasm/ muscle = yellow
- stained with picric acid ( small dye molecule )
collagen = red
- stained with acid fuschion ( larg dye molecule)
trichrome stain - usually
2 or more anionic dyes in conjunction with a heteropolyacid which result in selective coloring of collagen by one of the dyes
Heteropolyacids
Phosphomolybdic acid (PMA) &/or
Phosphotungstic acid (PTA)
- able to bind tissues from aqueous or alcoholic solutions
- colorless anionic dyes( may be demonstrated using UV or stannous chloride ) ( molybdenum or tungesten blue )
how PTA or PMA work
bind protiens & amino acids but NOT carbohydrates
COLLAGEN binds lots
cytoplasm binds smaller amounts
nuclei has little affinity ( doesn’t interfere with nuclear staining )
small dye is more easily replaced with PTA or PMA in collagen due to large pore size of collagen
cytoplasm & muscle staining with large molecule dyes is suppressed bc the cell membrane has small pores
small molecular dyes
PMA & PTA - there is considerable suppression of staining in all tissue components with small molecule anionic dyes ( heteropolyacids kick little molecules out of the collagen and hold that place for big molecule dyes)
ex of small dyes
Picric acid
Martius yellow
Eosin
Orange G
Biebrich scarlet
- will stain RBCs
big dye molecules
aniline blue
light green
acid fuchsin
- too big to be absorbed into small pores
factors affecting trichrome staining
- 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
nuclear stains
iron mordanted
resistant to low pH
- Weigerts
- Verhoeffs
- Celestine blue
acidic component = iron mordant
will be in combo with other stains in trichrome
fixation of trichrome stains
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
color of each component with trichrome staining
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)
Masson Trichrome stain
- 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 )
massons trichrome control & fixation
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)
Masson’s trichrome steps
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
Masson’s Trichrome Results
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)
liver Cirrhosis
Masson trichrome stain will be crowded with blue staining collagen
collagen takes over in liver cirrhosis
more fat than usual may also be seen
Gomori’s one step Trichrome steps
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)
Gomori’s results
nuclei- black
cytoplasm, keratin, muscle- red
Collagen & mucus - Blue
not best choice but most used for Muscle Biopsies *
Van Gieson stain
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
MSB stain ( Lendrum )
Martius Scarlet Blue
known for stianing…
dyes used
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
MSB stain results
Nuclei – Blue or Black
Muscle – Paler Red
Collagen – Blue
Fibrin – Red
RBCs – Yellow
Elastic fiber stains are used to demonstrate
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
Elastic fiber stains ( non specific )
Elastic fibers are stained but NOT specifically
H&E
HPS (hematoxylin phloxine saffron)
Congo Red
PAS
Elastic fiber staining ( more specific)
Specific or more intense stains include:
Verhoeff’s ( most widely used)
Aldehyde-fuchsin ( we use in lab)
Resorsin Fuchsin
Orcein (oldest)
elastin staining mechanisms ( less specific stains )
Eosin, Phloxine, or Congo Red stain
through ***columbic reaction between elastin protein and acid dyes
PAS stains ***glycoproteins in the fibers
elastin stain mechanisms ( more intense or specific staining )
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)
Verhoeff Elastic Stain colors of elastin
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
Verhoeffs elastic stain 3 stock solutions
Lugol Iodine (iodine, potassium iodide, H2O)
- iodine is mordant & oxidizers )
10 % ferric chloride
- mordant differentiator; Regressive Stain**
Alcoholic hematoxylin
Differentiation in Verhoeffs
- iodine remover & counterstain
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
Aldehyde Fuchsin elastic stain steps
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
Movat Pentachrome Russel mod
A combination of staining techniques are employed in one method
Stains mucin, fibrin, elastic fibers, muscle and collagen
Steps of Movat pentachrome russell mod.
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
Movat pentachrome russell mod.Results
Nuclei & Elastin…………….Black
Collagen…………………….Yellow
Ground substance & mucin….Blue
Fibrinoid, fibrin………Intense Red
Muscle………………………….Red
Metallic impregnation
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
metal empolyed by metallic impregnantion
Silver *** usually silver
Gold
Osmium
Mercury chromate( toxic)
Palladium
Lead
Copper
uses if metallic reduction
Reticular fibers
Bacteria (spirochetes)
Fungi
Neuropathology**
Detection of:
- Aldehydes
- Calcium
- Metal
points to remeber with matallic impregannation
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
oxidizer
sensitizer
bleach
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
stock solution
working solution
reducer
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)
toner
fixer
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
Argyrophil & Argentaffin
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
Gomori’s & Gordon & sweets steps
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
making the silver solution
This is usually the make or break part of silver impregnation
Involves silver nitrate and sodium or potassium hydroxide with ammonia
Preparation takes patience
Mallory PTAH demonstrates
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?
Mallory PTAH steps
Oxidize in permanganate
Bleach in oxalic acid
Stain overnight
Dehydrate rapidly
basement membrane stain
PAS-Methenamine Silver
Review method in book, note the use of microwave
Jones Methenamine Silver is more user
riendly
JMS
Similar to Grocott methenamine silver GMS
Hexamine and methenamine refer to the same substance
HEXAMETHYLENETETRAMINE
This substance replaces the ammonia & other alkali
lipid stain general points
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
lipid stains
Oil Red O and Sudan black B are commonly used (not water soluble)
Control not required
Mashing the coverslip may remove dye from tissues
uses of fat stains
Fat emboli (following bone fracture or crushing of fatty tissue)
Degenerating tissues
Identification of liposarcoma
Determine fat presence (stool, breast milk)
osmium tetraoxide
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
toludine blue
methylene blue
methyl green-pyronin Y
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
