What happens in heamatoxylin and eosin staining?
haematoxylin stains acidic structures purple
eosin stains basic structures pink
used to identify different types of cells and tissues
Advantages and disadvantages of immunohistochemistry staining?
Advantages - able to view tissue architecture, permanent
disadvantage - difficult to multiplex- stain more than one antigen
What happens in immunohistochemistry staining?
- fix tissue to preserve cell morphology and architecture
- antigen retrieval step to reverse harsh fixation and unmask epitopes
- can be used to diagnose neurodegenrative diseases and stages of cancer
What happens in immunofluorescence staining?
uses fluorescent instead of enzymatic detection
can use multiplexing
What happens in in-situ hybridisation?
uses a labelled nucleotide probe complementary to the target DNA instead of the primary antibody
can be used to asses chromosome integrity
can detect viral infection
What happens in phenocycler?
primary antibody have unique olgionucleotide barcodes, have a complementary oligonucleotide sequence with fluorescent label, used for specific for specific detection of each antibody
What happens in GeoMX digital spatial profiling?
- provides spatial context and quantitative expression of data
- antibody specific
- stain, select ROI, UV cleave, collect and dispense, count
What do continuous variables represent?
quantitative and scale variables, e.g weight, distance and temperature
What are ordinal variables?
variables with categories which can be ranked or ordered
What are nominal variables?
no quantitave factor, e.g gender, ethnicity, eye colour, blood type
What are the %s of normal distribution?
68% of values are within 1SD of the mean
95% of values are within 2SD
99.7% of values are within 3SD
What is flow cytometry? what can it do?
- technique used to analyse particles
- can count cells
- can measure cell size and granularity
- can detect fluorescent markers attached
What are the 3 things flow cytometry relies on?
- fluorescence
- antibodies that can be tagged with specific dyes and are specific
- advanced light detection and computing
What can flow cytometry be used for?
-Identifying and counting specific cell populations.
-Studying rare cells.
-Monitoring changes over time, such as:
-Immune responses
-Cell migration
-Cell proliferation
-Cell death.
What advanced techniques can be used to measure cells?
FACS (Fluorescence Activated Cell Sorting): sorts and collects specific cell populations.
Spectral flow cytometry: detects many fluorochromes simultaneously.
Mass cytometry: uses metal-tagged antibodies to measure even more markers.
Pros vs cons of flow cytometry:
Pros :
-Analyses millions of cells quickly.
-Detects rare populations.
-Provides detailed population-level data.
-Tracks changes over time.
Cons
-No information about cell location in tissue.
-Cells must be in suspension.
-Focuses on populations rather than individual cell context.
