Histological Methods Flashcards
define plane of section
3D structures may appear different when thin sectioned
describe types of sections
cross section (circle)
oblique (oblong)
longitudinal (long rectangle)
what type of section are most samples in
oblique section
why can’t you created a perfect sample (plane of section)
cannot orient sample when in wax
can’t move it after so what is seen is based on plane of section
what is the thickness of a section
5um
what is the diameter of a cell
10 um
what is the diameter of a lysosome
200nm
what is the thickness of PM
75A (7.5 nm)
describe H&E stain
hematoxylin and eosin
describe hematoxylin
basic
stains basophilic structures
blue or purple
name structures stained by hematoxylin
cell nucleus and other structures
basophilic
describe eosin
acidic
stains eosinophilic structures
pink
name structures stained by eosin
cytoplasm and collagen
what does basophilic mean (give ex of structures)
structures with nucleic acids
ribosomes
chromatin rich nucleus
cytoplasmic regions rich in RNA
what does eosinophilic mean (give ex of structures)
structures composed of intra/extra cellular protein
most of cytoplasm
T or F: structures have to be basic/acidic to be dyed a certain colour
FALSE
it’s based on affinity to dyes
describe electron microscope (how image is created)
Electron generated by cathode travels in high vacuum and hits photograph plate (phosphorescent screen)
what is an electron lucent structure
when electron passes through - so doesn’t hit anything
what is an electron dense structure
when electron hits something and is deflected away
describe resolving power of EM
2 dots come together
means that em can magnify way more than LM
LM = 1000x
EM = 160000x or 1.6 mil with cam zoom
what are the types of ways to look at specimens
light microscopy
electron microscopy
name the pathway to LM
chemical fixation
dehydration
clearing
infiltration
embedding
sectioning
mounting
removal of paragon
rehydration
staining
LM
OR
start with freezing —> sectioning and same ones down
describe freezing & down for LM
a way to preserve the specimen
then sectioning = done with cryostat
describe chemical fixation for LM
formalin
animal is anesthetized
what is an advantage of bouins fluid
better for H&E stain
what is perfusion and immersion
perfusion = inject into animal and works way inside out
immersion = take organ out and drop into fix and works way into tissue
want to stop decay and conserve ultrastructure
which method is faster: perfusion or immersion
perfusion is better
penetrates better and faster
describe dehydration for LM
dehydrate to put sample into blocks
need to get rid of water so increase concentration of ethanol (and decrease concentration of water)
want to use xylene to put tissue into wax blocks but water and xylene don’t mix
describe clearing LM
xylene is a paraffin solvent
describe infiltration LM
xylene/paraffin
adding dilute wax first (easier than adding to straight up wax)
describe embedding LM
putting in paraffin
describe sectioning LM
microtome
like a meat slicer
describe mounting LM
using a glass slide
describe removal of paraffin LM
using xylene to remove wax
describe rehydration LM
describe concentration of ethanol (increase water) so it becomes an AQ solution
describe staining LM
H&E and/or histochemical reaction
hematoxylin and eosin are water based so must first remove wax (removal of paraffin and rehydration)
describe chemical fixation EM
gluteraldehyde and osmium tetroxide = very fast fixative
preferably perfusion
describe dehydration EM
propylene glycol
doesn’t mix with water so must remove water
describe clearing EM
propylene glycol
epoxy solvent
describe infiltration EM
adding dilute plastic
propylene glycol/plastic
describe embedding EM
straight plastic
describe sectioning EM
ultramicrotome
very thin slices
describe mounting EM
copper grid
describe rehydration EM
there is NO REHYDRATION
plastic is fine to put on grid
describe staining EM
lead citrate and/or histochemical reaction
how does EM work
super thin slices go on grid and are blasted with electrons
describe what happens when electrons fly at grid/sample
electrons can go through and burn a whole = electron lucent structure
electrons can hit lead or grid and it takes off = electron dense structure
