Lecture 2 - staining

Question 1

why do we stain tissue

Answer 1

allows you to visualise its ultrastructure with more clarity

Question 2

whats another word for direct staining

Answer 2

histological staining

Question 3

what does staining actually do

Answer 3

colours an active component of the tissue

Direct interaction between a dye or a staining solution and the tissue e.g. Gomori trichome blue for skeletal muscle

Question 4

whats another word for indirect staining

Answer 4

histochemical staining

Question 5

what happenes in indirect staining

Answer 5

chemical reaction

links dye to tissue

link called mordant

Question 6

what does the chemical reation of indirect staining allow

Answer 6

localisation of specific substance

Question 7

what does immunohistochemical staining rely on

Answer 7

antibodies

Question 8

is Immunohistochemical staining direct or indirect

Answer 8

both

Question 9

what does Immunohistochemical staining allow

Answer 9

visualisation of specific molecules

Question 10

what is indirect staining in terms of antibodies

Answer 10

primary antibody that is unconjugated and then a secondary antibody that is conjugated

Question 11

what is direct staining in terms of antibodies

Answer 11

primary antibody that is fluorescently conjugated

Question 12

how many steps is indirect immunohistochemistry

Answer 12

2 steps

Question 13

how many steps is immunofluorescence

Answer 13

1 step

Question 14

factors that affect staining

Answer 14

quality of fixation

choice of fixative

time for stain

pH of staining

tissue thickness

Question 15

what happens if there is an overfix

Answer 15

may go in too much

blown apart

Question 16

what happens if the wrong fix is used

Answer 16

wont see what you want to

Question 17

what are the most common stains

Answer 17

• H and E stain in histopathology
• Gram stain in microbiology
• Romanowsky in haematology

Question 18

what is haematoxylin and eosin

Answer 18

haemotoxylin - basic dye, carries positive charge

eosin - acidic dye, carries negative charge

Question 19

what are basophilic and acidophilic

Answer 19

basophilic - acidic elements in tissue attracted to basic dyes

acidophilic - basic elements in tissue attracted to acidic dyes

Question 20

what does haemotoxylin bind to

Answer 20

structures like DNA

Question 21

what does eosin bind to

Answer 21

non DNA structures including proteins

Question 22

what does nuclei attract

Answer 22

haemotoxylin

stains dark blue

Question 23

what do cytoplasm, collagen and muscle attract

Answer 23

eosin

stains magenta

Question 24

what are the two types of staining

Answer 24

progressive

regressive

Question 25

what is differentiation of staining

Answer 25

removal of excess stain makes sure colour is retained only by wanted structures usually does with acidic alcohol

Question 26

whats the difference between muscle and connective tissue staining

Answer 26

muscle - deep pink connective tissue - pale pink

Question 27

what happens when the section of tissue isn't cut evenly

Answer 27

staining will be uneven better to use thin section of tissue patches of stains

Question 28

what is chattering

Answer 28

creates uneven parallel lines in the image following staining dye not applied enough

Question 29

can computer algortihsm remove chattering

Answer 29

yes, but unclear and distorted image is produced

Question 30

why is non-specific staining a problem

Answer 30

stain can sometimes stick to non-tissue elements on slide stain can bind elements of broken tissue generating an artificial signal

Question 31

how can you avoid non-specific staining

Answer 31

positive control -> perform staining on a well characterised tissue negative control -> tissue processed in the same way without the dyes

Question 32

how is non specific staining controlled in the lab

Answer 32

Every section of the lab has quality control logs as part of standard operating procedures (SOPS) External quality control lab participates in National External Quality Assurance Scheme Sends samples for testing once a month

Question 33

what do histopathologists do

Answer 33

Provides written report on surgical biopsy and cytology specimens Provides direct consultations on cases Contributes to equipment selection and technical methods Supports and contributes to postgraduate teaching