Histology

Question 1

What are the four main types of tissue?

Answer 1

Muscle Epithelial Nervous Connective (MEN with a C)

Question 2

Which complex structures does the ECM help form?

Answer 2

Collagen fibrils and basement membranes

Question 3

What are the functions of the extracellular matrix?

Answer 3

Mechanical support. Transport of nutrients to cell. Carry away metabolites and secretory products.

Question 4

What are the 5 stages of specimen preparation?

Answer 4

1) Fixation 2) Dehydration (alcohols) 3) Clearing (xylene) 4) Embedding (paraffin wax) 5) Sectioning

Question 5

Why is fixation necessary?

Answer 5

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.

Question 6

What are the different types of fixatives (group toxic and non-toxic)? (6)

Answer 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.

Question 7

Which fixatives are used for quick data?

Answer 7

Freezing or an alcoholic fixative (as it just coagulates proteins).

Question 8

What is the purpose of dehydration?

Answer 8

We need to remove water so that it can be embedded in paraffin wax as presence of water may mean it is immiscible.

Question 9

What are the advantages of using paraffin wax for embedding?

Answer 9

The paraffin wax needs to enter and solidify the structure for good sectioning. Melting point can be adjusted.

Question 10

When dehydrating, what can be used to do this and why is it used in a graded series?

Answer 10

Alcohol is used in a graded series to minimise tissue distortion.

Question 11

Why do we need the step ‘clearing’?

Answer 11

This is needed as the alcohol used for dehydration is not miscible in the paraffin wax, therefore, it has to be cleared.

Question 12

What are typical clearing agents? (7)

Answer 12

Zylene, Toluene, chloroform, benzene, petrol, histo-clear and histochoice.

Question 13

What is a microtome used for?

Answer 13

To section paraffin or resin embedded samples.

Question 14

How are frozen samples embedded and sectioned?

Answer 14

They are cyro embedded and the tissues are snap frozen in LN or dry ice (C02). The slides are then sectioned using a cyrostat.

Question 15

What has to be done if the stains are not compatible with paraffin wax?

Answer 15

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

Question 16

Describe the Haematoxylin and Eosin stain.

Answer 16

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.

Question 17

Describe the stain DAPI.

Answer 17

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).

Question 18

Describe the stain Alcian Blue.

Answer 18

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.

Question 19

Why do we not combine Alcian blue with H&E?

Answer 19

We need contrast in different components, alcian blue stains most things blue and H&E stains nucleus blue so there would be no contrast.

Question 20

Why should we not use the OIL RED O stain with a paraffin embedded section?

Answer 20

Because a paraffin embedded section will remove many fats and this stain specifically looks for fats, so it doesn’t make sense.

Question 21

Describe the stain Oil red O

Answer 21

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.

Question 22

Describe Millers Sirius Red

Answer 22

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.

Question 23

Describe masson’s trichrome

Answer 23

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.

Question 24

Describe PAS (periodic Acid solution)

Answer 24

Fuschin that reacts with aldehyde groups. Carbohydrates and carbohydrate rich macromolecules stain such as glycoproteins stain DEEP RED (MAGENTA). Nuclei stain BLUE.

Question 25

Describe the reticulin stain

Answer 25

Reduction of silver ions into silver metal and its decomposition onto reticulin fibers (mainly collagen type 3 FOUND IN LIVER). Reticulin fibers stains BLACK Collagen fibers stain BROWN Nuclei stain PINK.

Question 26

What are the 6 different types of connective tissue?

Answer 26

1. Bone
2. Cartilage (at the end of a bone)
3. Blood
4. Adipose
5. Fibrous (forms ligaments)
6. Loose (under the skin)

Question 27

What are the roles of connective tissue?

Answer 27

• Cartilage supports and binds tissues
• Stores nutritional substances e.g Ca stored in bones
• Produce protective and regulatory substances ECM

Question 28

What are the 3 layers of fasciae (connective tissue framework of the body)?

Answer 28

1. Superficial fascia: This is the uppermost layer, found between the skin and underlying organs, known as the subcutaneous layer/hypodermis. Contains areolar and adipose tissue.
2. Deep fascia (the middle layer) dense connective tissue
3. Subserous fascia: Found between deep fascia and serous membrane. Contains areolar tissue.

Question 29

What is the issue with the direct method used in immunohistochemistry?

Answer 29

It is not very sensitive.

Question 30

Describe the indirect method used for immunohistochemistry.

Answer 30

This is when an antibody marker is produced in a different species (e.g mice), specific to human antigens. Another labelled antibody is produced in another animal e.g. goat, that is specific to the FC region of the primary antibody.

Question 31

Why would you need to conduct antigen retrieval?

Answer 31

When you undergo tissue processing to examine it using microscopes, the step of fixation can cross-link antigens. This means they would be undetectable by antibodies.

Question 32

What are some methods for antigen retrieval? (4)

Answer 32

1. Enzymatic digestion
2. Citric acid
3. Heat
4. EDTA

ECHE, like ECHO for retrieval but extra e