How to examine cells and Tissue Flashcards

Question

How does immunohistochemistry work

Answer 1

- Tissues and cells present different proteins (antigens) on surface - Primary antibody binds to the antigen of interest - Secondary antibody detects primary antibody - Enzyme that is attached to the secondary antibody takes dye and converts it to a coloured product - coloured product stays with the enzyme at the desired area

Answer 2

- primary antibody attached to the protein on the tissue surface - a florescent tag is attached to antibody - light hits the tag forming a signal

Answer 3

* Surgical specimen on a metal disc frozen rapidly to –20 to –30°C - ‘Rock hard’ * The frozen specimen cut with microtome in cryostat in a freezer. * Stained with hematoxylin and eosin. * Sample preparation more rapid than with traditional histology technique

Answer 4

Paraffin wax formalin-fixed - fixed tissue - 24-48 hours to prepare - longer - permenant (lasts longer) - better clarity - Can detect pathological diagnosis Frozen Section - Fresh Tissue - 10 - 20 min - shorter - Months (Last LESS long) - Less Clear - Intraoperative Consulation ( eg check for cancer tissue during operation)

Answer 5

* Tissue Procurement * Tissue Processing: * Fixation & Preservation of tissue (with formalin to prevent putrefaction) OR * Frozen Section * Make sure that sample is very thin/transparent (2 to 20 μm) & small enough to fit the equipment * Embedding tissue (with paraffin wax) * Microtome * Tissue Staining (if applicable) * H&E and other staining methods

Answer 6

Light Microscope * Fix with Formalin * Embed in paraffin wax * Stain (e.g H&E/methylene blue)

Answer 7

Electron Microscope * Fix with Glutaraldehyde * Embed in Epoxy resin * Stain (e.g. Osmium tetroxide)

Answer 8

LIGHT microscope - Can view in natural colour - Larger field of view (2mm) diameter - Cheap and easy preparation - Can view living/ moving objects - magnification x600 - Resolution - 0.25uM Electron Microscope - Only monochrome images - Limited field of view ( 100uM) diameter - Difficult and expensive - Can only view dead or inert cells - Magnification - x500,000 - Resolution - 0.25 nm

Answer 9

Fix with Glutaraldehyde Embed in Epoxy resin Stain (e.g. Osmium tetroxide) can be viewed from an eyepiece directly

Answer 10

Fix with Glutaraldehyde Embed in Epoxy resin Stain (e.g. Osmium tetroxide) Use microtome with diamond knives the image goes to a collector

Answer 11

1. Laser excites a fluorescent dye and electrons are raised to higher energy level 2. As the electron relaxes back to its ground state (lower energy level), a light with higher wavelength emitted 3. Emitted light is sent through mirrors and a pinhole screen to a CMOS detector (like the one in your mobile phone’s camera)

Answer 12

* Because only one part of light (that bit that’s in focus) reaches the detector, the images are very sharp * Motorisation allows full section scanning * Means the entire depth of the cell/tissue can be examined

Answer 13

* ‘Cutting’ and ‘dicing’ * Collagenase and DNAse * Centrifugation steps on basis of cell density * Put cells into the appropriate growth medium * Culture cells * View under phase contrast microscope

Answer 14

* Allows manipulation of the cells and experiments to determine cells and thus tissue function

Answer 15

Advantages: * Absolute control over the physical environment * Homogeneity of sample * Reduced need for animal models Disadvantages: * Hard to maintain (skilled worker!) * Only grow small amount of tissue at high cost * Dedifferentiation * Instability, aneuploidy * 3 dimensional architecture is lost * Influence of other cells/tissues not maintained

Answer 16

* Very specialised technique used with living cells * It works by illuminating the sample with light that will not be collected by the objective lens and thus will not form part of the image * This produces the classic appearance of a dark, almost black, background with bright objects on it

Answer 17

1 millimeter (mm) = 1000 micrometers (μm) 1 micrometer (μm) = 1000 nanometers.

Answer 18

Tissue shrinkage may occur as the section undergoes alcohol dehydration. therefore might lead to decay In addition, during the embedding stage, the tissue may be compressed if the paraffin is too warm and undergo extra shrinkage during the cooling process. once removed from body, Organs tissue and cells no longer have protection of bodies immune system so can be digected by microbes.

Answer 19

Its dark background offers a high degree of contrast, making it easy to see samples on difficult backgrounds. This technique is easily accessible since many brightfield lab microscopes can be configured for darkfield illumination.

Answer 20

they can be studied in situ without the need for toxic and time-consuming staining processes.

Answer 21

This is because electons have a smaller wavelength that light thus they can reveal the tiny details