Samples and staining Flashcards
What does histology deal with?
Tissue (formed by cells)
What I tissue?
Discrete, organised cluster of cells having similar morphological characteristics
4 types of tissue
Epithelial
Muscular
Nervous
Connective
What is the standard microscope called?
Light transmission microscope
What do samples in light microscopes need to be?
Transparrent
How much can we magnify an object under the the light microscope?
1000x - 1500x
What field does a light microscope work in?
Bright field (whole sample is illuminated)
What kind of sample can be observed under light microscope?
Transparrent sample, sections of things like for example liver (often stained)
What is fixation?
Process that inactivates degradative enzymes and thus preserves cells and tissue structure
What is the first step to preparing a sample?
Fixation
What is the second step in preparing a sample?
Dehydration
What is dehydration?
Water of tissue is removed through a series of increasingly concentrated alcohol solutions (ending with 100% alcohol) which replaces water
What is often used in fixation?
Formaldehyde
What is the third step in preparing a sample?
Clearing
What is clearing?
Removing of alcohol in organic solvent in chive both alcohol and paraffin are miscible
What is the fourth (and most important) step in preparing a sample?
Infiltration
What is infiltration?
Allows substance (paraffin) to infiltrate tissue which creates a solid block which makes it easier to cut
What is used to cut samples?
Microtome (sliding or rotating)
What is the fifth step in preparing a sample?
Embedding
What happens after a sample is cut?
It is dried
What can you do if you need a sample looked at under a microscope quickly?
Snap freezing (in liquid nitrogen)
What is staining useful for?
Being able to see the sample clearly and decode/understand what we’re looking at
Most important and popular staining
Hematoxylin Eosin (H&E)
With H&E what color does cell nuclei stain?
Blue
With H&E what color does most components of the cell cytoplasm stain?
Pink/red
How does hematoxylin get its color?
It is oxidated and sorted with a metal salt which turn it into a color compound used for staining
What does it mean that nuclei are basophilic structures, they’re basophiles?
They bind well to basic compounds
It’s an acids
What does nuclei stain well with?
Something that is positively charged, basic
Why do nuclei stain well with something that is positively charged?
Because they are basophilic
What is the cytoplasm of a cell?
Eosinophilic
What does it mean that the cytoplasm of a cell is acidophilic/eosinophilic?
Binds well to acids
What is the PAS method of staining used for?
Staining carbohydrates and glycoproteins
How many dyes does Masson’s trichrome use?
3
What color does Masson’s trichrome stain kreatin and muscle fibers?
Red
What color does Masson’s trichrome stain collagen and bone?
Blue
What color does Masson’s trichrome stain cytoplasm?
Light red or pink
What color does Masson’s trichrome stain cell nuclei?
Dark brown
What I the May-Grünwald-giemsa mainly used for?
Smear preparations of blood and bone marrow cells
Method of staining to get most precise idea of location and structure of cells
Immunochemistry/immunohistochemistry
What does immunochemistry staining rely on?
The use of antibodies to highlight the precedes of specific targets in a sample
What is the direct immunochemistry?
Antibodies that have been previously functionalized with the molecules that allow us to detect it with a fluorescent light or an enzyme
What is the antibody that is directed against the antigen in the sample called?
Primary antibody
What is the primary antibody in the indirect approach?
Just a simple antibody without any labelling
What is the primary antibody in the direct approach?
Antibody labeled with for example fluorescent light
What are polyclonal antibodies?
Several clones of antibodies for all the different epitopes are made for that antigen
Increases likelihood of staining because even if one epitope is destroyed in processing there are antibodies fitting for the others
What are monoclonal antibodies?
One type of antibody is isolated and it fits to one kind of epitope
Where do antibodies attach to antigens?
Epitopes
What can monoclonal antibodies do that is useful?
They can sometimes differentiate between conformational state of the protein
How are antibodies labeled?
Attached to another molecule (conjugation)
Example of molecules/things used for conjugation/labelling of antibodies?
Biotin
HRP (enzyme)
Fluorophore
Magnetic bead
Agarose
What is needed to observe samples stained with fluorophores?
Microscope equipped for fluorescence (immunofluorescence). Light with filter which selects/only allows passage of that specific wavelenght
Can a fluorescent sample be observed even if it is not transparrent?
Theoretically yes, the light doesn’t go through it, it is reflected (adept fluorescence)
What do you need to do/add when using enzymes to label antibodies?
Add substrate/chemical compound that can be attacked by the enzyme and turns colourful
What is bright field microscopy?
No staining or fixation, only the two pigment cells can be seen
What is phase-contrast microscopy?
Often used with living cells. Structures with different refractive indices affect in-phase and produce an image of these features in all the cells
Special filters added to microscope
What phenomenon does phase-contrast microscopy use?
Destructive and constructive interference
How does dark field microscopy work?
Deviate the light rays in such a way that they would not reach the eye piece
What is confocal microscopy?
A technique to increase precision of image you’re looking at
What is polarising microscopy?
Polarisers (2 sets) select one orientation of the light which the light which goes through. Some light goes through both polarisers and that is then what is visible.
How does a stereomicroscope work?
Light source from the top illuminates the sample off of which the light is reflected
What is the limit of the resolution of a light transmission microscope?
0.2 micrometers/ 200 nanometers
What are the two main kinds of electron microscopes?
Transmission electron microscope, the tem
Scanning electron microscope, the sem
Do the electrons pass through the sample in an electron microscope?
Yes, through and to the detector which convert the electron beam into an image
What does the TEM microscope function like?
Light microscope but with electrons
What does the SEM microscope function like?
Stereo microscope, the electron beam is reflected by the sample
Which electron microscope gives a 3d image of the surface?
SEM/scanning electron microscope
You don’t see the inside but get the outside in great detail
What is used to stain a sample when using a TEM microscope?
Specific metal salts
What is the sample coated with when using a SEM microscope?
Metal, for example gold
