tissues Flashcards
(120 cards)
What is tissue, cellular and sub-cellular structure?
- tissues = how cells combine with EC material and each other to form tissue
- cellular = cell shape and organisation of components to support function
- sub-cellular = organelles
What are the four main tissue types in the body?
- connective
- nervous
- muscle
- epithelial
What are the two main components of tissue?
- cells
- ECM (highly-organised molecules formed by cells to influence complex structure)
What are the two functions of the ECM?
- mechanical support
- nutrients and metabolite exchange
What are the five stages of preparing a specimen for microscopy?
(SPSSM)
- specimen dissection
- preparation
- sectioning
- staining
- microscopy
(16-48 hrs)
What is the aim of tissue processing?
to embed tissue in medium firm enough to support and enable cutting of thin sections
Summarise the main stages of tissue processing and the purposes of each one.
FDCESC
(Hint - Frances Does Crazy Expeditions So Cleverly)
- fixation: treated with chemical agent to prevent autolysis and shape changes i.e. aldehydes and ketones
- Dehydration: fixative + water removed from specimen and replaced with dehydration fluid (graded series from 10-100% ethanol)
- Clearing: dehydrating fluid replaced with miscible fluid (dehydrating fluid and embedding medium) using clearing agents i.e. zylene, tulouene, chloroform, benzene, petrol, histo-clear, histo-choice
- Embedding: tissues embedded with medium to provide support during sectioning i.e. plastic resin, paraffin wax (3 μm), polymerising resin
- Sectioning: cut using microtome, sectioned with cryostat (>6μm)
- Cryo-embedding: tissue samples snap frozen in LN₂/CO₂ and slides stored
Why is paraffin wax used for embedding?
- polycrystalline, elastic mixture with high MP
- increases hardness, improves ribboning and adhesion between specimen and wax
What are the three requirements for tissues after embedding stage?
- no clearing agent
- no dust particles
- rapidly-cooled to reduce wax crystal size
What is staining?
- permits examination of tissues by LM
- tissues and cells are translucent (>1 stain can be used)
What are most stains not compatible with?
paraffin wax
How are slides readied for examination?
CRSDCMC
- cleared in xylene
- rehydrated via (graded) alcohol series + washed
- stained
- dehydrated
- cleared with xylene
- mounted using xylene medium + coverslip
- cryo-embedded and stored
For each stain state the structures and the colours they are stained:
a) H and E
b) alcian blue
c) oil red o
d) masson’s trichrome
e) reticulin
f) periodic acid solution (PAS)
g) DAPI (4′,6-diamidino-2-phenylindole)
h) millers sirius red
a)
- (H) nucleus and proteins → blue
- (E) cytoplasm → pink
b)
- acid mucins + proteoglycans → blue
- nuclei → red/black
c)
- fat → brilliant red
- nuclei → blue
d)
- nuclei and basophilic structures → blue
- cytoplasm, muscle, erythrocytes, keratin → bright red
- collagen → green/blue
e)
- reticulin fibers → black
- collagen fibers → brown
- nuclei → pink
f)
- glycoproteins → magenta
- nuclei → blue
g)
- DNA and nuclei → blue
h)
- elastin → dark purple/black
- collagen → red/pink
- phase contrast → changes brightness for clarity
- collagen fibers → befringent (shadowy)
What is IHC?
- application of specific antibodies to tissue preparation
- for localization of specific antigens
What type of detection system is IHC and how is the antibody visualised?
- highly-sensitive
- using marker (i.e. fluorescent dye, colloidal gold)
What is the direct method of IHC?
- single-step
- use of directly-labelled primary antibody supplied to detect antigens within tissue
What is the indirect method of IHC?
- utilizes unlabeled primary antibody and secondary labelled antibody
- binds to it to detect tissue antigen
What is IHC antigen retreival?
- tissue antigens can be masked by tissue fixation
- different methods can be used to reveal antigen: enzymatic digestion, citric acid, EDTA, heat
What can be used for inhibition of endogenous tissue components (reactions during staining) and for the blocking of non-specific sites?
(HInt - sounds like Alvin)
- inhibition of endogenous tissue: 3% (v/v) H₂O₂ and 0.01 % (w/v) Avidin
- blocking of non-specific sites: 10% (v/v) normal serum
State some controls which can be used in IHC.
- positive control - tissue known to contain epitope
- diluent control - buffer only (tissue section known not to express target antigen)
- omission of primary antibody
- omission of secondary antibody
- isotype antibody - isotype of antibody used to bind to same antigen (see if it binds/not)
Give examples of IHC
- collagen type IV: collagenfound in basal lamina
- α-Gal; carbohydrate in mammalian cell membranes
- fluorescent examples: SM cells, DAPI and actin, human skin, E-cadherin
State pros and cons of IHC.
pros:
- high specificity for molecular species
- can be used for light, confocal, or electron microscopy
cons:
- time-consuming and expensive
- fixation can interfere with Ab (Alcian Blue) binding particularly with small molecules
- reproducibility - false positives due to cross-reactivity
- qualitative
What are the five types of microscopy.
CDKPF
- bright field/kohler illumination
- phase contrast
- differential interference contrast (DIC) microscopy
- fluorescence
- confocal
State the pros and cons of confocal microscopy.
pros: - excellent resolution in thick samples - greater number of flurophore as specific λs used to illuminate samples - collects light from a single focal plane cons: - photo-bleaching and phototoxicity - increased sensitivity to noise - technical method - labour-intensive