Pre Lab

Question 1

What is immunohistochemistry (IHC)?

Answer 1

A technique that uses antibodies to detect specific antigens in tissue sections.

Question 2

What is the aim of this practical?

Answer 2

To detect glucagon in rat pancreas tissue.

Question 3

What type of molecule is glucagon?

Answer 3

A 29–amino acid peptide hormone.

Question 4

What does glucagon do?

Answer 4

Raises blood glucose by stimulating glycogen breakdown in liver.

Question 5

Which pancreatic cells secrete glucagon?

Answer 5

Alpha (α) cells.

Question 6

Which pancreatic cells secrete insulin?

Answer 6

Beta (β) cells.

Question 7

Which cells secrete somatostatin?

Answer 7

Delta (δ) cells.

Question 8

What are the two functional parts of the pancreas?

Answer 8

Exocrine and endocrine.

Question 9

What does the exocrine pancreas do?

Answer 9

Secretes digestive enzymes via ducts.

Question 10

What does the endocrine pancreas do?

Answer 10

Releases hormones into bloodstream.

Question 11

What are pancreatic islets?

Answer 11

Clusters of endocrine cells inside pancreas.

Question 12

What surrounds pancreatic islets?

Answer 12

Exocrine acini.

Question 13

What controls pancreatic hormone secretion?

Answer 13

Blood glucose levels directly.

Question 14

Is pancreas controlled by pituitary?

Answer 14

No.

Question 15

How thick are the tissue sections used?

Answer 15

5 µm.

Question 16

Why label the frosted slide side?

Answer 16

To identify sample.

Question 17

Why use pencil instead of pen?

Answer 17

Ink dissolves during staining.

Question 18

What is an antigen?

Answer 18

A molecule recognized by an antibody.

Question 19

What is the antigen in this experiment?

Answer 19

Glucagon.

Question 20

What is the primary antibody?

Answer 20

Mouse antibody that binds glucagon.

Question 21

What does the secondary antibody bind?

Answer 21

The primary antibody.

Question 22

Why is a secondary antibody used?

Answer 22

Signal amplification + detection.

Question 23

What molecule is attached to the secondary antibody?

Answer 23

Biotin.

Question 24

What binds biotin?

Answer 24

Streptavidin.

Question 25

What enzyme is attached to streptavidin?

Answer 25

Horseradish peroxidase (HRP)

Question 26

What does ABC stand for?

Answer 26

Avidin/Biotin Complex.

Question 27

Why use the ABC method?

Answer 27

To amplify signal and increase sensitivity.

Question 28

What is the order of staining steps?

Answer 28

Primary → Secondary → Streptavidin-HRP → Chromogen.

Question 29

Why can't we see antibody binding directly?

Answer 29

Antibody complexes are colourless.

Question 30

What substrate is added for colour development?

Answer 30

AEC + hydrogen peroxide.

Question 31

What does HRP do?

Answer 31

Catalyses chromogen reaction.

Question 32

What colour indicates glucagon presence?

Answer 32

Red.

Question 33

Why is the reaction product useful?

Answer 33

It is insoluble and stays at antigen site.

Question 34

What does red staining indicate?

Answer 34

Location of glucagon-producing cells.

Question 35

Where would you expect staining in pancreas?

Answer 35

Alpha cells in islets.

Question 36

What is digital pathology?

Answer 36

Scanning slides to analyse/share electronically.

Question 37

Advantage of digital slides?

Answer 37

Remote collaboration + storage + analysis.

Question 38

Why use secondary antibodies instead of labelled primary?

Answer 38

Amplifies signal + flexible detection system.

Question 39

What ensures signal amplification in ABC method?

Answer 39

Multiple biotin molecules bind streptavidin complexes.

Question 40

What determines stain location?

Answer 40

Antigen-antibody binding site.

Question 41

Why must chromogen product be insoluble?

Answer 41

So it stays at antigen location.

Question 42

What would happen if HRP was missing?

Answer 42

No colour reaction.

Question 43

Explain immunostaining principle in one sentence.

Answer 43

Specific antibodies bind target proteins and enzyme reactions create coloured deposits showing their location in tissue.