Immunohistochemistry & Microscopy Flashcards
what is immunohistochemistry?
a technique used to detect specific antigens (proteins) in tissue sections using labelled antibodies
briefly describe the process of immunohistochemistry
- tissue is fixed and sectioned
- primary antibody binds to target antigen
- secondary antibody (often enzyme-conjugated) binds to the primary antibody
- substrate reacts with the enzyme to produce a visible colour change (or fluorescence)
intensity/degree of colour change is proportional to the amount of presence of the specific protein we want to detect
what is a primary antibody?
primary antibodies directly bind bind to the specific target antigen
what is a secondary antibody?
secondary antibodies bind to primary antibodies - often conjugated with enzymes (e.g., horseradish peroxidase) or fluorophores to amplify detection
what is immunocytochemistry?
a technique used to detect specific antigens (proteins) on individual cells using labelled antibodies
briefly describe the process of immunocytochemistry
- cells are fixed and sectioned
- primary antibody binds to target antigen on cells
- secondary antibody (often enzyme-conjugated) binds to the primary antibody
- substrate reacts with the enzyme to produce a visible colour change (or fluorescence)
what are polyclonal antibodies?
antibodies produced by multiple B-cell clones - recognise multiple epitopes on an antigen
(epitope - specific part of an antigen an antibody binds to)
what are monoclonal antibodies?
produced from a single B-cell clone, recognise one specific epitope
compare polyclonal & monoclonal antibodies
- polyclonal abs are produced by multiple B-cell clones & recognise multiple epitopes on an antigen
- monoclonal antibodies are produced by a single B-cell clone & recognise one specific epitope
- polyclonal abs are more sensitive & less specific
between monoclonal and polyclonal antibodies, which ones are more specific but less sensitive? why?
monoclonal antibodies
MORE SPECIFIC: only recognise one epitope on an antigen; reduces cross-reactivity & makes them highly specific
LESS SENSITIVE: only bind to one epitope, so may not detect antigens with slight variations or low abundance, making the overall signal weaker compared to polyclonal antibodies
what is widefield fluorescence microscopy?
imaging method where the entire sample is illuminated simultaneously using UV or visible light - fluorophores in the sample absorb this light and emit fluorescence which is detected by the microscope’s camera
briefly describe how widefield fluorescence microscopy works
- sample is labelled with fluorescent dyes or tagged proteins (e.g. GFP)
- broad-spectrum light source excites the fluorophores
- emitted florescence is passed through a filter to remove unwanted wavelengths & the final image is captured using a digital camera
what is confocal microscopy?
imaging technique that uses a laser and a pinhole aperture to focus light on a specific plane within a sample - generates high-resolution 3D images
briefly describe how confocal microscopy works
- laser beam scans the sample & excites fluorophores in a focused spot
- emitted fluorescence passes through a pinhole aperture = blocks out-of-focus light
- detected fluorescence is used to construct an image point by point using a computer
- multiple layers can be scanned to build a 3D reconstruction of the sample
compare widefield fluorescence & confocal microscopy
widefield fluorescence:
- illuminates the entre sample at once, best for large and thin samples
- has a lower resolution due to out-of-focus light but is faster
confocal:
- focuses laser beam on a thin plane, best for thick & 3D samples
- higher resolution but slower
how does fluorescence microscopy rely on Stokes shift?
fluorophore absorbs high-energy light at a shorter wavelength, loses some energy as heat from molecular vibrations, then releases the remaining energy as light at a longer wavelength
the emitted light is filtered and captured to generate an image
Stokes shift allows for:
- separation of excitation & emission light, preventing signal overlap
- allows multicolour imaging with different fluorophores
what is light sheet microscopy/ selective plane illumination microscopy?
imaging technique using a thin sheet of light to illuminate an entire plane at once - allows for high-resolution 3D imaging of whole specimens
DAPI is a common fluorescent dye used to label and visualise different cellular components. how does it interact with its sample for fluorescent visualisation?
DAPI intercalates between DNA bases, makes the DNA fluoresce blue under UV light
what colour does Sytox-Orange fluoresce DNA?
red (binds to DNA)
how does phalloidin work as a fluorescent dye?
binds to filamentous actin- labels cytoskeletal structures
examples of fluorescent dyes?
- phalloidin (binds to filamentous actin, labels cytoskeletal structures)
- DAPI (binds to DNA - fluoresces blue)
- Sytox-Orange (binds to DNA, fluoresces red)
- GFP (fluoresces green, used in live imaging)
what is flow cytometry?
technique that detects & measures the physical and chemical characteristics of individual cells in a fluid suspension
briefly describe how flow cytometry works
- cells are stained with fluorescent markers specific to proteins of interest
- a laser illuminates each cell as it passes through a narrow stream (single-file)
- detectors measure fluorescence intensity and light scattering - provide info on: cell size, granularity, protein expression levels
- data is analysed and different cell populations are characterised
what is the significance of forward & side scatter in flow cytometry?
forward scatter: measures cell size (larger cells = more forward scatter)
side scatter: measures cell granularity (e.g. granules in different immune cells) (more granularity = more side scatter)
