Chapter 6 - Fixation Flashcards
whereby the chemical constituent of the fixative is taken in and becomes part of the tissue by forming cross-links or molecular complexes and giving stability to the protein.
additive fixation
examples of additive fixation
formalin
mercury
osmium tetroxide
whereby the fixing agent is not incorporated into the tissue, but alters the tissue composition and stabilizes the tissue by removing the bound water attached to H-bonds of certain groups within the protein molecule.
non-additive fixation
Specimens should be transferred to fixative quickly ______ after surgery as deterioration will commence with the loss of blood supply.
<1hour
Tissues should be fixed in a sufficient volume of solution; generally in a ratio of _____ or at least _____ fixative to specimen, for penetration to occur in the most efficient manner.
20:1 or at least 10:1
______ to fixation must be removed or incised (e.g. fascia, bone, feces, thick tissue)
anatomical barriers
large specimens must be sectioned or inflated with fixative (e.g. ____)
lung
opened and cleaned (________) to allow penetration.
gastrointestinal tract
Pinning specimens to a corkboard or inserting a ____ or _____ into tubular structures can improve fixation and reduce tissue distortion.
paper or gauze “wick”
The volume of fixative is important. Traditionally, the amount of fixative used has been _______ the volume of tissue to be fixed.
10-20 times
Hydrogen Ion Concentration: Fixation is best carried out close to neutral pH, in the range of ___
6-8
Common buffers
phosphate, bicarbonate, cacodylate, and veronal
Commercial formalin is buffered with phosphate at a pH of __
7
Temperature:
Many laboratories use tissue processors that work at ___ for regular tissue processing
40C
For electron microscopy and some histochemistry, the ideal temperature is ____
0-4C
Formalin heated to ____ is sometimes used for the rapid fixation of very urgent biopsy specimens, although the risk of tissue distortion is increased
60C
Thickness of section:
Tissue blocks should be small (e.g. ______ for electron microscopy and _____ wide for light microscopy) and thin (no more than ____ for light microscopy)
1 to 2mm^2 - EM
2cm^2 wide - LM
0.4cm - LM
Large solid tissue, such as _____, should be opened or sliced thinly to improve penetration of fixatives.
uterus
Brain is usually suspended whole in ___ buffered formalin for 2-3 weeks to ensure fixation and some hardening prior to sectioning.
10%
For solid material (e.g., liver) the longest dimension should not exceed ______
10-15mm
The best results are usually obtained using ________ (400-450 mOsm; isotonic solutions are 340 mOsm).
slightly hypertonic solutions
_____ is commonly added to osmium tetroxide fixatives for electron microscopy.
sucrose
Formaldehyde is normally used as a ___ solution
10%
glutaraldehyde is normally used as a ___ solution
3%
Primary fixation in buffered formalin is usually carried out for ______
2-6 hours
Most of the formalin can be washed out after fixation for ____
24 hours
For electron microscopy, it is recommended that diced tissues be fixed for _____
3 hours
There are four major groups of fixatives, namely the:
the aldehydes, oxidizing agents, alcohol based fixatives and the metallic group of fixatives
2 types of aldehydes
formaldehyde
glutaraldehyde
2 types of oxidizing agents
osmium tetroxide
potassium permanganate
acts by cross-linking proteins
oxidizing agents
alcohol based fixatives
methyl alcohol, ethyl alcohol, acetic acid
these are protein-denaturing agents
alcohol based fixatives
_____ act by forming insoluble metallic precipitates like mercuric chloride and picric acid.
metallic fixatives
The choice of the fixative is based on _______
tissue and anticipated ancillary tests
______ are made up of only one component substance.
simple fixatives
2 types of metallic fixatives
mercuric chloride
chromate fixatives
these are the simple fixatives
Aldehydes
a. Formaldehyde
b. Glutaraldehyde
Metallic Fixatives
a. Mercuric chloride
b. Chromate fixatives
Picric acid
Acetic acid
Acetone
Alcohol
Osmium Tetroxide
are those that are made up of two or more fixatives which have been added together to obtain the optimal combined effect of their individual actions upon the cells and tissue constituents.
compound fixatives
are those that permit the general microscopic study of tissue structures without altering the structural pattern and normal intercellular relationship of the tissues in question.
microanatomical fixatives
7 types of microanatomical fixatives
10% formal saline
10% neutral buffered formalin Heidenhain’s Susa
Formal sublimate (formal corrosive)
Zenker ‘s solution
Zenker-formal (Kelly ‘s solution)
Bouin’s solution
Brasil’s solution
are those that preserve specific parts and particular microscopic elements of the cell itself.
cytological fixatives
are those that preserve the nuclear structures (e.g., chromosomes) in particular. They usually contain glacial acetic acid as their primary component due to its affinity for nuclear chromatin.
nuclear fixatives
pH of nuclear fixatives
pH of 4.6 or less
5 types of nuclear fixatives
Flemming’s fluid
Carnoy’s fluid
Bouin’s fluid
Newcomer’s fluid
Heidenhain’s Susa
______ has been found to react with viruses, and causes the loss of their infective power.
mercuric chloride
are those that preserve cytoplasmic structures in particular. They must never contain glacial acetic acid which destroys mitochondria and Golgi bodies of the cytoplasm.
cytoplasmic fixatives
pH of cytoplasmic fixatives
pH of more than 4.6
5 types of cytoplasmic fixatives
Flemming’s fluid without acetic acid
Kelly’s fluid
Formalin with “post-chroming”
Regaud ‘s fluid (Muller ‘s fluid)
Orth ‘s fluid
For RNA, the precipitant fixatives - ________ - give the best quantitative results using frozen tissues as the standard.
ethanol and acetone
are those that preserve the chemical constituents of cells and tissues.
histochemical fixatives
4 types of histochemical fixatives
Formal Saline 10%
Absolute Ethyl Alcohol
Acetone
Newcomer’s Fluid
______ is the process of placing an already fixed tissue in a second fixative in order
secondary fixation
______ may be done before dehydration and on deparaffinized sections before staining, usually with 10% formalin or 10% formol saline as a primary fixative.
secondary fixation
tissue fixed in 10% buffered neutral formalin may require secondary fixation with ________ (that acts as a mordant)
zenker’s fixation
stain used for connective tissue
masson’s trichrome
stain used for collagen
mallory’s aniline blue stain
stain used for striated muscle
phosphotungstic acid- hematoxylin (PTAH)
is a form of secondary fixation whereby a primarily fixed tissue is placed in aqueous solution of 2.5-3% potassium dichromate for 24 hours to act as mordant for better staining effects and to aid in cytologic preservation of tissues.
post-chromatization
is the process of removing excess fixative from the tissue after fixation in order to improve staining and remove artefacts from the tissues. Several solutions may be used.
washing out
Tap water is used to remove:
a. excess chromates from tissues fixed in Kelly’s, Zenker’s, and Flemming’s solutions
b. excess formalin
c. excess osmic acid
used to wash out excess amount of picric acid (Bouin’s solution).
50-70% alcohol
is used to remove excessive mercuric fixatives.
alcoholic iodine
may be found in surgical
specimens particularly in liver biopsies, associated with an intense eosinophilic staining at the center of the tissue in H&E stained sections.
crush artifact
In conventional histopathological
techniques, lipids are largely removed
during preparation of tissues.
lipid fixation
_______ or _______ should be used
for demonstrating lipid in tissues,
followed by a general lipid stain.
cryostat or frozen sections
Fixatives containing ______ and ______ can be effective for preservation of lipids in cryostat sections.
mercuric chloride and potassium dichromate
may be used to preserve phospholipids.
baker’s formal-calcium
Carbohydrates are hydrophilic; they hold much water in the extracellular space by
hydrogen bonding.
carbohydrate fixation
________ is a better
fixative in human skin compared with neutral buffered formaldehyde.
alcoholic formaldehyde
The most useful fixatives for preserving glycogen are alcohol-based, such as:
Rossman’s fluid or cold absolute alcohol
There is better retention of glycogen if the section is coated with ______
celloidin
the most commonly used fixatives for amino acid histochemistry.
Neutral buffered formal saline or formaldehyde vapor
_____, _____, and ______ with the whole procedure performed at 4°C.
osmium tetroxide, glutaraldehyde and paraformaldehyde
For electron histochemistry and electron immunocytochemistry, ________ is useful
Karnovsky’s paraformaldehyde-glutaraldehyde
FIXATION FOR ENZYME HISTOCHEMISTRY
4% formaldehyde or formal saline overnight
pathology for the demonstration of
various antibodies.
FIXATION FOR IMMUNOFLUORESCENCE
FIXATION FOR IMMUNOFLUORESCENCE:
In many instances, _____ and _____ may be used.
formalin-fixed and paraffin embedded sections
In more sensitive cases, the tissue must be prepared as a cryostat section and fixation limited to a few seconds in:
absolute methanol or acetone
To ensure free access of the antibody to its antigen, the cells must be fixed and permeabilized.
FIXATION FOR IMMUNOHISTOCHEMISTRY
_______ such as alcohols
and acetone remove lipids and dehydrate the cells, while precipitating the proteins on the cellular architecture.
organic solvents
_______ (such as paraformaldehyde) form intermolecular bridges, normally through free amino groups, thus creating a network of linked antigens.
CROSS-LINKING REAGENTS
Fix cells in -20°C acetone for 5-10 minutes. No permeabilization step needed following acetone fixation.
acetone fixation
Fix cells in -20°C methanol for 5-10 minutes. Permeabilization step is needed following methanol fixation.
methanol fixation
Fix cells in cooled 95% ethanol, 5% glacial acetic acid for 5-10 minutes.
ethanol fixation
Fix in cooled methanol, 10 minutes at –20 °C.
Remove excess methanol.
Permeabilize with cooled acetone for 1 minute at –20 °C.
methanol-acetone fixation
1:1 methanol and acetone mixture. Make the mixture fresh and fix cells at -20 C for 5- 10 minutes.
Methanol-Acetone Mix Fixation
1:1 methanol and ethanol mixture. Make the mixture fresh and fix cells at -20 C for 5- 10 minutes.
methanol-ethanol mix fixation
Fix cells in 10% neutral buffered formalin for 5-10 minutes. Rinse briefly with PBS. Permeabilize with 0.5% Triton X-100 for 10 minutes.
Formalin Fixation
Fix in 3-4% paraformaldehyde for 10-20 minutes. Rinse briefly with PBS. Permeabilize with 0.5% Triton X-100 for 10 minutes.
Paraformaldehyde-Triton Fixation
Fix in 4% paraformaldehyde for 10-20 minutes. Rinse briefly with PBS. Permeabilize with cooled methanol for 5-10 minutes at –20 °C.
Paraformaldehyde-Methanol Fixation
