Tissue analysis how-to
How to make a slide from a tissue specimen
-process -stains
How to tell what you’re looking at
-looking at an unfamiliar slide
3-D reconstruction from 2-D sections
Differentiating between basic tissue types
-stuff that looks like other stuff
HOW IS A SLIDE MADE?
1) obtain tissue block (biopsy, cadaver)
2) fix – chemical treatment – usually aldehydes -harden soft tissue, coagulate protein – crosslinks form -stop enzymes working -stop small molecules diffusing away, anchor carbohydrates -stop bacterial decomposition
3) embed – stiffen tissue block for cutting sections -dehydrate tissue, replace water with solvents -solvents replaced with waxes, plastics → solidify
4) cut embedded (or frozen) block →sections – 5 – 50 um thick for light microscope (50 – 150 nm thick for electron microscope)
5) stain sections - increase contrast - tissue structures visible
5) mount sections on glass slides
Stains
Why stain? -fresh sections colorless, no contrast
Visualize specific tissue components with selective stains -can’t show all components in any one tissue section
Components of tissues visible by staining:
-acidic components – use basic dyes: hematoxylin, toluidine blue, methylene blue -basic components – use acidic dyes: eosin -carbohydrates – use periodic acid-Schiff (PAS), stains mucus, basement membranes -proteins - use H & E, picrosirius, trichrome stains – collagen fibres -lipids - use lipid-soluble stains: osmium, Sudan black, oil red O
Combine stains visualize multiple tissue elements at once -hematoxylin and eosin (H & E): both acidic and basic components -trichromes – three stains together
Hematoxylin and eosin – most common stain
Hematoxylin – basic dye stains acidic tissue components blue/purple/black
acidic components attract basic dyes = basophilic
shows: nucleic acids: nuclei, nucleoli, ribosomes acidic parts of glycoproteins, glycosaminoglycans hyaline cartilage matrix, loose connective tissue matrix
Eosin – acidic dye stains basic tissue components red/pink
basic components attract acidic dyes = acidophilic
shows: collagen, mitochondria, secretory granules cell cytoplasm
H & E STAINING OF ILEUM H8 H8 H & E STAINING OF ILEUM H8 H8 H & E STAINED COLON H9 H & E DUODENUM
H63 H & E STAINED COLLAGEN IN DUODENUM SUBMUCOSA
H63 H & E STAINED DUODENUM MUSCULARIS EXTERNA
H63 H & E STAINED AXILLA H68 H & E AXILLA GLAND SECRETORY REGION H68 H & E STAINED COLLAGEN IN AXILLA RETICULAR DERMIS
H68 H & E STAINED PAROTID GLAND H66
serous-secreting acini mucus-secreting acini Periodic acid - Schiff stain - many carbohydrates
2 steps: -periodic acid oxidises adjacent -OH groups on hexoses → aldehydes form
-Schiff's reagent (fuschine) stains aldehydes
Sugars stain pink-purple:
basement membrane glycoproteins
mucopolysaccharides (goblet cells)
PAS STAIN OF BASEMENT MEMBRANES slide 136 STAINING PROTEINS IN EXTRACELLULAR MATRIX
TYPE I COLLAGEN FIBRES H & E (pinkish, variable) picrosirius (and polarized light) trichrome stains Mallory's, Masson's
RETICULAR FIBRES (TYPE III COLLAGEN) silver stains
ELASTIN Verhoff's elastin stain orcein aldehyde fuscin H & E STAINED COLLAGEN IN AXILLA RETICULAR DERMIS
H68 TRICHROME STAINS
- 3 (or more) dyes: differentiate tissue elements visually
- aniline blue stains collagen blue
- acidic dyes: cytoplasm, muscle - pink-red-purple erythrocytes - orange -yellow nuclei - brown-black KIDNEY - MALLORY'S TRICHROME - collagen blue H201 OVIDUCT - trichrome H51 OVIDUCT - trichrome H51 URETER - trichrome H13 URETER - trichrome H13 URETER - trichrome H13 SPLEEN - silver stain: reticular fibres H60 AORTA - Verhoff's elastin stain H88 AORTA TUNICA MEDIA - Verhoff's elastin stain H88 EPIGLOTTIS
H & E H28
Verhoff's elastin stain H29 EPIGLOTTIS - Verhoff's stain, elastin fibres in cartilage H29 LIPID STAINS
-lipid-soluble oil red O Sudan black osmium MYELIN STAINED WITH OSMIUM - spread segment of peripheral nerve H24 Tissue analysis how-to
How to make a slide from a tissue specimen
-process -stains
How to tell what you’re looking at
-looking at an unfamiliar slide
3-D reconstruction from 2-D sections
Differentiating between basic tissue types
-stuff that looks like other stuff
Looking at a new slide
Examine by eye first, then at lower higher magnification
-Don’t try to guess what it is yet!
Ask: -how cellular is the tissue (nuclei: how many, how big)? -how are the cells organized? How large? Where are their nuclei? -how much extracellular matrix is there? -are there fibres in the matrix? what color? how dense? how organized? -are there holes in the tissue? How many? How big? Are any connected?
Then ask what you can recognize: (does anything look familiar?) -overall appearance of the tissue? -basic tissue types? -cell types? -fibre types? -staining patterns in extracellular matrix?
Use what you know to work out what you don’t know
What do the instructions say to look for?
Look it up in the text (ask the instructors!)
19-13 Cormack 1-4 Cormack 1-8 Cormack 1-9 Basic Histology 1-30 Basic Histology 18-15 The “orange” problem
Ross & Pawlina 1-11 The “orange” problem
Cormack 1-10 Look-alike tissues
Telford Plate 1 Look-alike tissues
stratified squamous epithelium, thin skin
transitional epithelium, bladder
Telford Plate 1 Look-alike tissues
Telford Plate 4 Look-alike tissues
cardiac muscle
skeletal muscle
Telford Plate 4 Look-alike tissues
Telford Plate 6 Look-alike tissues
whole nerve, longitudinal section
dense irregular connective tissue, dermis of skin
Telford Plate 6