Lecture 2 - staining Flashcards
why do we stain tissue
allows you to visualise its ultrastructure with more clarity
whats another word for direct staining
histological staining
what does staining actually do
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
whats another word for indirect staining
histochemical staining
what happenes in indirect staining
chemical reaction
links dye to tissue
link called mordant
what does the chemical reation of indirect staining allow
localisation of specific substance
what does immunohistochemical staining rely on
antibodies
is Immunohistochemical staining direct or indirect
both
what does Immunohistochemical staining allow
visualisation of specific molecules
what is indirect staining in terms of antibodies
primary antibody that is unconjugated and then a secondary antibody that is conjugated
what is direct staining in terms of antibodies
primary antibody that is fluorescently conjugated
how many steps is indirect immunohistochemistry
2 steps
how many steps is immunofluorescence
1 step
factors that affect staining
quality of fixation
choice of fixative
time for stain
pH of staining
tissue thickness
what happens if there is an overfix
may go in too much
blown apart
what happens if the wrong fix is used
wont see what you want to
what are the most common stains
- H and E stain in histopathology
- Gram stain in microbiology
- Romanowsky in haematology
what is haematoxylin and eosin
haemotoxylin - basic dye, carries positive charge
eosin - acidic dye, carries negative charge
what are basophilic and acidophilic
basophilic - acidic elements in tissue attracted to basic dyes
acidophilic - basic elements in tissue attracted to acidic dyes
what does haemotoxylin bind to
structures like DNA
what does eosin bind to
non DNA structures including proteins
what does nuclei attract
haemotoxylin
stains dark blue
what do cytoplasm, collagen and muscle attract
eosin
stains magenta
what are the two types of staining
progressive
regressive
what is differentiation of staining
removal of excess stain
makes sure colour is retained only by wanted structures
usually does with acidic alcohol
whats the difference between muscle and connective tissue staining
muscle - deep pink
connective tissue - pale pink
what happens when the section of tissue isn’t cut evenly
staining will be uneven
better to use thin section of tissue
patches of stains
what is chattering
creates uneven parallel lines in the image following staining
dye not applied enough
can computer algortihsm remove chattering
yes, but unclear and distorted image is produced
why is non-specific staining a problem
stain can sometimes stick to non-tissue elements on slide
stain can bind elements of broken tissue generating an artificial signal
how can you avoid non-specific staining
positive control -> perform staining on a well characterised tissue
negative control -> tissue processed in the same way without the dyes
how is non specific staining controlled in the lab
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
what do histopathologists do
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
