IHC Flashcards
What is the purpose of IHC
To diagnose tumors whether they are benign or malignant
-to classfy them - origin, primary or metastatic
prognosis
therapy - chemo, radiation or drugs
diagnose infections like h pylori
immunofluorescence
AB bound to dye
-use frozen unfixed tissue
Enzyme immunohistochemistry
Enzyme + AB (horseradish peroxidase, alkaline phosphatase)
add substrate or chromogen to visualize AG sites
MONOCLONAL
Epitope specific
Less sensitive
Very pure, more expensive
POLYCLONAL
Reacts with many epitopes
Less specific
More sensitive
Less pure, less expensive
step one
epitope/Antigen Retrieval
Purpose and method
purpose
to break crosslinks formed during fixation so tissue is exposed
Heat-induced epitope retrieval (HIER) (2) Enzyme-induced epitope retrieval (EIER)
What is Epitope Retrieval - HIER
temp
solution
example
High heat
Solutions: Sodium citrate buffer or EDTA
Examples: modified pressure cooker, laboratory microwave oven, vegetable streamer, circulating water bath
Epitope Retrieval - EIER
what is it
example
Proteolytic enzyme
Examples: pronase, ficin, protease, pepsin
step 2
blocking
what types of reactions and how many
2 reactions
1-3% H2O2 to block tissue endogenous peroxidase activity. E.g. tissues that contain many RBC- causes background staining
2-Antibody attachment to high charged collagen & connective tissues.
step 3
staining methods
direct
Substrate+chromogen+Enzyme HRP+labelled AB+AG = visualized
indirect -Avidin-Biotin Complex (ABC)
ABC bound to 2ndary AB conjugated with biotin through biotinylation + primary unlabeled AB
step 4 detection system
with chromogen which is an electron that gets oxidized and provides a color to AB+AG reaction
3,3’-diaminobenzidine tetrahydrochloride
DAB
Dark brown, granular reaction at antigen site
if your enzyme is HRP
and your chromogens are DAB and silver
what colors do you see
1- brown
2-black
if your enzyme is AP
and your chromogens are Fast red and NBT/BCIP
what colors do you see
1- fuschia
2-blue
Types of staining
nuclear
membranous
cytoplasmic
Pre-analytical Factors affecting Staining: Fixation
-↑ cold ischemic time - ↓ antigen stability
-Fixation time
-Fixative used
-Temperature and pH of fixative (high temps can damage AG sites)
Vimentin staining used to check for adequate fixation
Pre-analytical factors affecting staining: processing
-adequate dehydration and was infiltration is good for IHC staining
-high temps during processing can damage antigen sites
Pre-analytical Factors affecting staining: Microtomy
-Waterbath should contain clean deionized water
-Gloves worn to avoid squamous cell contamination
-Charged slides (+)
-Section thickness: 3-4µ
-Centre the tissue section on the slide
must use artifact free sections
CONTROLS
how are they done
positive
-need to be run with EACH AB stain EACH time its performed
-multi tissue control
-do on same slide as pt specimen
Negative
-dont use primary AB
