fixation lec

fixation

The first and most critical step in histotechnology

autolysis or putrefaction

fixation prevents

lysosomal enzymes

The fixatives employed prevent autolysis by inactivating __

preserve the morphologic and chemical integrity of the cell in as life-like a manner as possible

primary goal of fixation

harden and protect the tissue from the trauma of further handling

second goal of fixation

autolysis

__ results from tissue digestion by intracellular enzymes that are released when organelle membranes rupture

bacterial decomposition

putrefaction aka

putrefaction

__ brought about by microorganisms which may already be present in the specimen

20% -30%

During processing, however, the specimen may shrink and lose how many percent of volume

hypertonic solution

cell shrinkage is due to

hypotonic solution

cell swelling is due to

Postmortem decomposition ("autolysis")

occurs due to the action of these hydrolytic enzymes

freeze drying

Cryopreservation, usually in the form of

Chemical fixation

usually achieved by immersing the specimen in the fixative solution

additive fixation, non additive fixation

two basic mechanisms involved in fixation

Additive fixation

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.

Non-additive fixation

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

agitation

will also enhance fixation of the specimen.

insufficient ratio of tissue volume to fixative volume

The most common error in histotechnology is

6-8

Fixation is best carried out close to neutral pH, in the range __

formalin-heme pigment

Acidity favors formation of

formalin-heme pigment

black, polarizable deposits in tissue

7

Commercial formalin is buffered with phosphate at a pH of

40C

Many laboratories use tissue processors that work at

0-4C

For electron microscopy and some histochemistry, the ideal temperature is

Refrigeration

used to slow down decomposition if the tissue needs to be photographed and cannot be fixed immediately

1 to 2 mm2

tissue block size for electron micro

0.4 cm

tissue block size for light microscopy

formalin and alcohol

what penetrates the best

glutaraldehyde

what penetrates the worst

2 cm2 , and no more than 4 mm. thick

the recommended size of the tissue is

10-15mm

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

shrink

If cells are fixed in a hypertonic solution, the cells may

swell

. If the cells are fixed in a hypotonic solution, the cells may

glutaraldehyde (0.25%)

an ideal concentration for immuno-electron microscopy.

heat, vacuum, agitation or microwave

Fixation time can be cut down by using

2-6 hours

Primary fixation in buffered formalin is usually carried out for

3 hours

For electron microscopy, it is recommended that diced tissues be fixed for

60°C

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

Simple Fixatives

fixatives that are made up of only one component substance

aldehydes, metallic fixatives, picric acid, acetic a, acetone, alco, osmium tetroxide

simple fixatives main example

a. Formaldehyde b. Glutaraldehyde

aldehydes example

a. Mercuric chloride b. Chromate fixatives

Metallic Fixatives example

compound fixatives

fixatives that - 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.

Microanatomical Fixatives

fixatives that -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.

cytological fixatives

fixatives that are those that preserve specific parts and particular microscopic elements of the cell itself

nuclear fixatives

fixatives that - are those that preserve the nuclear structures (e.g., chromosomes) in particula

glacial acetic acid

primary component of nuclear fixatives

4.6 or less

ph of nuclear fixatives

mercuric chloride

found to react with viruses, and causes the loss of their infective power.

Kelly's sol

Zenker-formal AKA

cytoplasmic fixatives

fixatives that - are those that preserve cytoplasmic structures in particular. They must never contain glacial acetic acid which destroys mitochondria and Golgi bodies of the cytoplasmcyt

more than 4.6

ph of cytiplasmic fixativer fixatives

precipitant fixatives

For RNA. __ give the best quantitative results using frozen tissues as the standard

histochemical fixatives

are those that preserve the chemical constituents of cells and tissues.

secondary fixation

the process of placing an already fixed tissue in a second fixative in order

post chromatization

the process of placing an already fixed tissue in a second fixative in order

washing out

the process of removing excess fixative from the tissue after fixation in order to improve staining and remove artefacts from the tissues.

tap water

what is used to wash out formalin, osmic acid, kellys, zenkers, flemming

50-70% alcohol

what is used to wash out excess amount of picric acid

alcohol iodine

what is used to remove excessive mercuric acid

5 mm

Tissues should not be more than

Formalin pigment

well-known artifact that may be produced under acid conditions.

crush artifact

" 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

cryostat

__ should be used for demonstrating lipid in tissues, followed by a general lipid stain

alcohol fixatives

generally recommended for glycogen fixation.

Neutral buffered formal saline or formaldehyde vapor

the most commonly used fixatives for amino acid histochemistry

osmium tetroxide, glutaraldehyde and paraformaldehyde

The most useful primary fixatives for electron microscopy

Karnovsky's paraformaldehyde-glutaral-dehyde

For electron histochemistry and electron immunocytochemistry,

Immunofluorescence techniques

commonly used in pathology for the demonstration of various antibodies.

formalin-fixed and paraffin

what is used in immunofluorescence

organic solvents

alcohols and acetone remove lipids and dehydrate the cells, while precipitating the proteins on the cellular architecture.

cross linking reagents

form intermolecular bridges, normally through free amino groups, thus creating a network of linked antigens

acetone fixation

Fix cells in -20°C, 5-10 mins

methanol fixation

Fix cells in -20°C, 5-10 mins, permeabilization needed

Ethanol fixation

Fix cells in cooled 95% ethanol, 5% glacial acetic acid for 5-10 minutes.

Methanol-Acetone 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 Mix Fixation

1:1 methanol and acetone mixture. Make the mixture fresh and fix cells at -20 C for 5-10 minutes.

Methanol-Ethanol Mix Fixation

1:1 methanol and ethanol mixture. Make the mixture fresh and fix cells at -20 C for 5-10 minutes.

Formalin 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.

Paraformaldehyde-Triton 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-Methanol Fixation

Fix in 4% paraformaldehyde for 10-20 minutes. Rinse briefly with PBS. Permeabilize with cooled methanol for 5-10 minutes at -20 °C.

Antigen Retrieval

methods in immunohistochemistry have shown that some of the reactions of fixation are reversible, particularly those of formaldehyde, but there is considerable variation in the quality of antigen preservation with various agents.

heat induced epitope retrieval

'HIER' meaning

37°C and 45°C

commonly employed temp

microwave fixation

allows light microscopic techniques used in routine histopathology to be performed adequately

microwave fixation"

Fresh tissue, placed in saline or other isotonic solution, can be irradiated to produce primary fixation

microwave-assisted fixation

microwaved to assist the fixative action of the fixing agent

2 mm , 70% ethanol

After microwaving they should immediately be sliced to __and placed in __

Crosslinking Fixatives

act by creating covalent chemical bonds between proteins in tissue. This anchors soluble proteins to the cytoskeleton, and lends additional rigidity to the tissue

Precipitating (or denaturing) fixatives

that act by reducing the solubility of protein molecules and (often) by disrupting the hydrophobic interactions that give many proteins their tertiary structure.

formaldehyde

By far the most commonly used fixative in histology is

3.7%-4.0%

percentage of formaldehyde in phosphate buffered saline

Formaldehyde

a gas produced by the oxidation of methyl alcohol, and is soluble in water to the extent of 37-40% weight in volume

10% neutral buffered formalin (NBF, approximately 4% formaldehyde)

The most widely used fixative for routine histology is

Karnovsky's paraformaldehyde-glutaraldehyde solution

The best known mixture of fixative is

Acrolein

another aldehyde which has been introduced as a mixture with glutaraldehyde or formaldehyde

Acrolein

. It penetrates tissues rapidly, preserves morphology and enzyme activity at low concentrations, and may be used for immersion fixation of surgical biopsies

12 - 24 hours

10% Formal-Saline Fixation time

10% Formal-Saline

This is a simple microanatomical fixative made up of saturated formaldehyde (40%, by weight volume) diluted to 10% with sodium chloride. This mixture of formaldehyde in isotonic saline was widely used for routine histopathology prior to the introduction of phosphate buffered formalin.