Imaging Flashcards

Question

What is specificity in IF and IHC staining?

Answer 1

Specificity - degree to which antibody only binds to protein of interest. Determined mainly by the primary antibody - only bind to antigen nothing else. Tissue quality will also effect specificity.

Answer 2

Lower limit of detection, the minimum amount of antigen that can be detected. Determined by primary antibody and tissue quality, and the detection system used.

Answer 3

Indirect detection - primary antibody added then fluorescent labelled secondary antibody binds, multiple binding so amplification of signal. Direct - fluorochrome is conjugated to primary antibody. More simple but less sensitive. Can be made more sensitive by amplification by detection system.

Answer 4

A lectin that binds to a-linked fucose residues in glycoproteins and glycolipids in blood. This is a marker for endothelial cells. It is not an antibody but is directly conjugated to a fluorophore, so is easy to combine with a range of different antibodies, without worrying about species specificities of secondary antibodies.

Answer 5

A fluorescent stain that binds strongly to DNA in the nucleus. So it is a good positive control as it shows where the cells are.

Answer 6

Uses a labelled nucleotide probe complementary to the target DNA instead of primary antibody. Once bound, can detect using chromogenic detection (ISH) or fluorescent detection (FISH).

Answer 7

DNA is denatured to give single strand that the complementary labelled probe can bind to. Then add primary antibody specific to the label. Then secondary antibody with enzyme attached to convert chromogen to visual precipitate or fluorescent label.

Answer 8

Assess chromosome integrity Detect viral infection DNA-FISH can be multiplexed.

Answer 9

Detect gene expression Very useful in short-lived or secreted proteins (hard to visualise).

Answer 10

A more advanced method of RNA fluorescent in situ hybridisation. Can multiplex, has high specificity and sensitivity - as 2 probes are bound and it is amplified.

Answer 11

Complementary probe binds, then second complementary probe binds adjacent to first. When the two probes are bound together, then pre-amplifier can bind, then amplifier, then labelled probe.

Answer 12

IHC/ISH can be used to visualised 1-3 proteins or genes. IF/FISH can be used to visualise 4-6 proteins or genes. It is limited by the number of different species for primary and secondary antibodies. Also limited to the number of different fluorochromes that can be visualised together.

Answer 13

Sequential immunofluorescence (seqIF). Has successive cycles of staining, imagining, then elution. Two antigens are imaged per cycle. It is fast, uniform and reproducible staining.

Answer 14

Can combine with RNAscope to analyse both protein and RNA, spatial multi-omics Analyse 40+ targets.

Answer 15

Use primary antibodies with unique oligonucleotide barcode attached Add complementary oligonucleotide sequences to the barcodes, have fluorescent dyes, allows specific detection of each antibody. Iterative imagining cycles - stain, reveal 3 antibodies, image, remove, repeat, until all proteins in section are imaged.

Answer 16

Stain up to 3 proteins using fluorescent labelled antibodies which are morphology Markers - used to decide which parts of the tissue to look at - the regions of interest. It is then DNA stained to identify it. To examine RNA, add probes specific to the RNA wanted to analyse.

Answer 17

Probes have UV-photocleavable oligonucleotide barcode attached, they're cleaved by UV light. For proteins, add antibodies specific to the proteins, have oligonucleotide barcode attached that is UV photocleavable. Only those with the probes or antibodies attached will be cleaved.

Answer 18

UV light is shone onto the regions of interest. Only the oligonucleotide barcodes from antibodies that are bound by proteins are cleaved. The barcodes are collected and dispensed into a 96-well plate, then counted using NGS or an integrated system.

Answer 19

It provides spatial context - where the region is from, and quantitative expression data - the level of gene/protein expression in the region.

Answer 20

Quantify gene expression in regions but there are multiple cell types Want to look at specific cells, so can use Xenium platform, CosMx, phenocycler-fusion.

Answer 21

Hybridisation of probes Assembling of flow cell Can then be analysed by interactive data analysis at a single cell level.

Answer 22

DNA/RNA-ISH /FISH are least complex and expensive. Then RNAscope, then spatial transcriptomics then single cell transcriptomics.

Answer 23

Least: IHC IF Multiplex IF Spatial proteomics Single cell proteomics.

Answer 24

Tissue or tumour is embedded in agarose gel for support. Sliced used vibratome, thicker sections of live tissue. Produces tumour slice for analysis and imaging Can be cultured and kept alive for a week.

Answer 25

Treated with drugs or antibodies for checkpoint inhibitors Culture under different conditions e.g. low oxygen and see effects Add immune cells to see behaviour Killing assay if tumour cells, see if immune cells can kill tumour cells.

Answer 26

Can stain using limited numbers of markers. Takes layers and stacks together to produce 3d live image. Then can fix and embed cultures for higher plex imaging.