Chapter 6 - Fixation Flashcards by Deleted Deleted

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

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examples of additive fixation

formalin
mercury
osmium tetroxide

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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

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Specimens should be transferred to fixative quickly ______ after surgery as deterioration will commence with the loss of blood supply.

<1hour

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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

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______ to fixation must be removed or incised (e.g. fascia, bone, feces, thick tissue)

anatomical barriers

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large specimens must be sectioned or inflated with fixative (e.g. ____)

lung

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opened and cleaned (________) to allow penetration.

gastrointestinal tract

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Pinning specimens to a corkboard or inserting a ____ or _____ into tubular structures can improve fixation and reduce tissue distortion.

paper or gauze “wick”

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The volume of fixative is important. Traditionally, the amount of fixative used has been _______ the volume of tissue to be fixed.

10-20 times

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Hydrogen Ion Concentration: Fixation is best carried out close to neutral pH, in the range of ___

6-8

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Common buffers

phosphate, bicarbonate, cacodylate, and veronal

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Commercial formalin is buffered with phosphate at a pH of __

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Temperature:
Many laboratories use tissue processors that work at ___ for regular tissue processing

40C

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For electron microscopy and some histochemistry, the ideal temperature is ____

0-4C

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Formalin heated to ____ is sometimes used for the rapid fixation of very urgent biopsy specimens, although the risk of tissue distortion is increased

60C

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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

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Large solid tissue, such as _____, should be opened or sliced thinly to improve penetration of fixatives.

uterus

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Brain is usually suspended whole in ___ buffered formalin for 2-3 weeks to ensure fixation and some hardening prior to sectioning.

10%

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For solid material (e.g., liver) the longest dimension should not exceed ______

10-15mm

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The best results are usually obtained using ________ (400-450 mOsm; isotonic solutions are 340 mOsm).

slightly hypertonic solutions

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_____ is commonly added to osmium tetroxide fixatives for electron microscopy.

sucrose

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Formaldehyde is normally used as a ___ solution

10%

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glutaraldehyde is normally used as a ___ solution

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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