Histology Flashcards
(32 cards)
What are the four main types of tissue?
Muscle Epithelial Nervous Connective (MEN with a C)
Which complex structures does the ECM help form?
Collagen fibrils and basement membranes
What are the functions of the extracellular matrix?
Mechanical support. Transport of nutrients to cell. Carry away metabolites and secretory products.
What are the 5 stages of specimen preparation?
1) Fixation 2) Dehydration (alcohols) 3) Clearing (xylene) 4) Embedding (paraffin wax) 5) Sectioning
Why is fixation necessary?
1) To prevent autolysis/degradation and bacterial attack 2) Stop enzymatic activity 3) Allow the tissue to be as close as possible to living state i.e. no molecules lost. 4) Prevents it taking up any fluids used to prevent a change in volume/shape.
What are the different types of fixatives (group toxic and non-toxic)? (6)
1) Toxic fixatives: Aldehyde, ketones (i.e. acetone), Alcohol (i.e. methanol) and heavy metals (i.e osmium tetroxide). 2) Non-toxic: Zinc fixative and freezing.
Which fixatives are used for quick data?
Freezing or an alcoholic fixative (as it just coagulates proteins).
What is the purpose of dehydration?
We need to remove water so that it can be embedded in paraffin wax as presence of water may mean it is immiscible.
What are the advantages of using paraffin wax for embedding?
The paraffin wax needs to enter and solidify the structure for good sectioning. Melting point can be adjusted.
When dehydrating, what can be used to do this and why is it used in a graded series?
Alcohol is used in a graded series to minimise tissue distortion.
Why do we need the step ‘clearing’?
This is needed as the alcohol used for dehydration is not miscible in the paraffin wax, therefore, it has to be cleared.
What are typical clearing agents? (7)
Zylene, Toluene, chloroform, benzene, petrol, histo-clear and histochoice.
What is a microtome used for?
To section paraffin or resin embedded samples.
How are frozen samples embedded and sectioned?
They are cyro embedded and the tissues are snap frozen in LN or dry ice (C02). The slides are then sectioned using a cyrostat.
What has to be done if the stains are not compatible with paraffin wax?
1) Slides cleared in xylene 2) Rehydrated through alcohol series 3) Washed in water to remove the alcohol 4) Stained 5) dehydrated 6) cleared in xylene 7) Mounted using xylene medium and glass coverslip
Describe the Haematoxylin and Eosin stain.
This is also known as a H&E stain. Think of the order of positive and negative, this reflects on the H&E as, H is cationic/+ve and E is anionic/-ve. The positive H, binds to negative parts of a cell, i.e. the nucleus, which is stained BLUE and the cytoplasm is PINK.
Describe the stain DAPI.
Fluorescent stain, Binds to A-T rich regions in the minor groove of the DNA, Pass through intact cell membrane, Used to stain both live and fixed cells Stains nuclei BLUE. (Think about DAPI is a rapper who fluoresces, he dances with his rich mATes doing the minor groove).
Describe the stain Alcian Blue.
A mucin stain that stains mucins, proteoglycans and cartilage BLUE. Nuclei is stained RED/BLACK. (REMEMBER MC P unjab) Think about it as it doesn’t do what the name blatantly suggests and alcian blue is underestimated and thought to be a sheep and stain the nucleus blue, but no, it beats it red/black.
Why do we not combine Alcian blue with H&E?
We need contrast in different components, alcian blue stains most things blue and H&E stains nucleus blue so there would be no contrast.
Why should we not use the OIL RED O stain with a paraffin embedded section?
Because a paraffin embedded section will remove many fats and this stain specifically looks for fats, so it doesn’t make sense.
Describe the stain Oil red O
Used to stain fat in unfixed frozen sections (cyro embedded) as processing may remove fat content from cells and tissues. Stains fats BRILLIANT RED and nuclei BLUE. Think of it like this stains fats in unfit/unfixed people/sections. Processing/running will remove fat content from cells and tissues.
Describe Millers Sirius Red
This can be used with a combination of both kohler illumination (normal light microscopy) and phase contrast (polarising filters). Under kohler, elastin stains DARK PURPLE/BLACK, collagen stains RED/PINK and under phase contrast collagen fibers are birefringent (different colours, mainly the neo collagen (new collagen) which helps recognise if there are new cells/tumours). Remember this as miller a boy is serious about red riding hood, he has a elastic band and when he thinks about her he pulls it making his skin go black/dark purple. When he sees her, his skin/collagen goes pink/red but when he sees her from a contrast glass his collagen is bifringent.
Describe masson’s trichrome
Used to stain connective tissue. Nuclei and basophilic (base-liking) stained BLUE. Cytoplasm, muscle, erythrocytes and keratin BRIGHT-RED. Collagen is stained GREEN OR BLUE. REMEMBER MASSON IN MECKA.
Describe PAS (periodic Acid solution)
Fuschin that reacts with aldehyde groups. Carbohydrates and carbohydrate rich macromolecules stain such as glycoproteins stain DEEP RED (MAGENTA). Nuclei stain BLUE.
