L6-Imaging, more on IHC,IF Flashcards
(27 cards)
What does imaging allows?
-detailed view/visualise organisms, tissues, cells or cellular components
1.Which cell/cell type did the DNA/RNA/protein originate from? - single-cell seq.
2.Where were those cells located in the tissue?
3.What structures/cells/extracellular proteins/etc. were nearby?
Imaging approaches provide what?
spatial context - detailed view of everything.
What do you want to image?
-Thin sections of preserved tissue
>formalin-fixed paraffin-embedded (FFPE) tissue
>fresh-frozen tissue
-cell monolayer
>cell suspension – cytospin or dot cells onto slide
>adherent cells – culture on coverslip
whole animal > zebrafish, mouse
humans
Why are we using +vely charged slides in imaging?
To help attract and hold onto negatively charged molecules, like DNA or proteins, making it easier to see them under a microscope.
Name different types of approaches to imaging tissues, cells or cellular components.
-H&E staining
-Immunohistochemistry staining
-Immunofluorescence staining
-In situ DNA hybridisation/RNAscope
-Multiplex spatial proteomics/transcriptomics
>COMET
>PhenoCycler
>GeoMx Digital Spatial Profiling
-Single cell spatial multi-omics
Define Haematoxylin and Eosin (H&E) Staining
-Haematoxylin stains acidic structures (e.g. DNA) blue/purple – nuclei
-Eosin stains basic structures (e.g. ribosomes) pink – cytoplasm
-H&E stain is used to diagnose cancer
More details on IHC staining
-Fix tissue (if not already fixed) – to preserve cell morphology and tissue architecture
-Antigen retrieval (using heat or enzymes) – to reverse effects of ‘harsh’ fixation and ‘unmask’ epitopes
-Permeabilise (if staining for intracellular antigens) – to allow antibodies to enter cells
-Block using e.g. serum or BSA – to reduce background or non-specific staining
Controls in IHC staining
Positive – sample known to include antigen of interest
Negative – sample that does not include antigen of interest
Isotype control – antibody of same isotype as 1o but not specific for antigen of interest
Name pros and cons for IHC staining
😊: simple workflow, can view tissue architecture, standard brightfield microscopy, permanent
😔: difficult to multiplex (stain for more than one antigen)
Uses of IHC staining in medicine
-diagnosis of neurodegenerative diseases e.g. Parkinson’s and Alzheimer’s disease
-diagnosis and staging of cancer, and prediction of treatment response
Define immunotherapy
Antibodies that block PD-L1 – PD-1 interaction (and reactivate anti-tumour immune responses) are used in cancer treatment = immunotherapy
How to select patients for immunotherapy?
PD-L1 IHC is an approved test to select patients for immunotherapy.
More details on the IF staining
-Basic IF is similar to IHC but uses fluorescent instead of enzymatic detection.
Pros and cons of IF staining
😊:allows multiplexing using 1o antibodies raised in different species and 2o antibodies conjugated to different fluorochromes
😔: requires precise technique, it’s time-consuming, and sometimes it might give non-specific binding, leading to potential misinterpretations.
What are the 2 important considerations for IHC/IF staining
Specificity
and
sensitivity
Define Specificity
Specificity – degree to which antibody only binds protein of interest.
>primary antibody – key determinant
>tissue quality
Define sensitivity
Sensitivity – lower limit of detection, minimum amount of antigen that can be detected
>Multiple secondary antibodies can bind to a single primary antibody so there will be amplification.
Define DNA In Situ Hybridisation (DNA-ISH/DNA-FISH) and how to detect ?
-Uses a labelled nucleotide probe complementary to the target DNA instead of 1o antibody
-Detection can use chromogenic (ISH) or fluorescent (FISH) techniques
Uses of DNA hybridisation
-assess chromosome integrity >checking whether the DNA strands can properly bind to each other, indicating that the DNA is intact and not damaged. This process helps determine if there are mutations, structural abnormalities, or breaks in the chromosome.
-detect viral infection
Define RNAscope
-RNA Fluorescent In Situ Hybridisation (RNA-FISH)
-detect gene expression – particularly useful for short-lived or secreted proteins
-RNA SCOPE for protein that short-lived and also wider visualising gene expression.
What do we use probes for in imaging ?
-Complimentary probe binds and a secondary c.probe binds adjacent to it.
-2 probes indicate that they have high specificity.
Define COMET
-Sequential immunofluorescence (seqIF)
>successive cycles of staining-imaging-elution
>two antigens imaged/cycle
-Use of chip enables “Fast fluidic exchange”
→ fast, uniform and reproducible staining
-Can combine seqIF and RNA scope to analyse both protein and RNA → Spatial multi-omics
Analyse 40+ targets
-Microfluidic chip gives a control environment for imaging.
Define Phenocycler
-1o antibodies have unique oligonucleotide barcode
-complementary oligonucleotide sequences to the barcodes are attached to fluorescent dyes, allowing specific detection of each antibody
-iterative imaging cycles
-reveal 3 antibodies at a time
Define GeoMx Digital Spatial Profiling
-provides both spatial context and quantitative expression data
-analyse 570+proteins and or whole transcriptome.
-Can stain up to 3 proteins
-decodes which tissue we want to look at
-markers help to select the areas.
- Stain using UV-photocleavable oligo fluroscent antibodies
- Select ROI- region of interest
- UV-cleave
