Tissue Preservation Flashcards
What is tissue preservation ?
The process in which a tissue is kept alive outside the organism
Why is it done ?
Its done to investigate the tissue find out if there are issues with it or any association with that particular tissue .As well as look for changes .
When should fixation be carried out ?
Should be carried out soon as possible after the removal of the tissues due to case of surgical pathology or soon after death soon after death with autopsy to prevent autolysis.
What are the five types of fixative types ?
Aldehydes Picrate Alcohol Oxidizing agents Mercurial
They all have similar functions which needs to be destroyed .
What are the steps o tissue fixations ?
- Isolate the tissue into cold PBS as soon as possible .
- Wash tissue with PBS to remove all blood
- Place tissue in fixative for 10-15 minutes to one hour .
- Cut tissues to proper size .The size can be 2X2 mm to 1X2 but thickness must be thinner than 3 mm for better fixation . The cutting surface of the tissue .
- Transfer the tissue to fixative and swirl the container to ensure all tissues are completely immersed in fixative .However, the volume must be 20-30 time tissue volume
- Fix tissues at 40 over night
- Check sample the next day to ensure proper fixation .
What happens after Fixation ?
- Wash with PBS 1 min
- Wash with PBS 30 MIN X40
- Dehydrate :
30% ethanol in ddh20 for 2hrs
50 ethanol in ddh20 for 2hrs
70% ethanol in ddh20 for 4hrs
95% ethanol for 3hours or 0/N
100% ethanol for 1 hour x2 Store at -20 C 100% ethanol for 1 hour x3 at Rt Xylene 30-40 min x2 , Check embryos 5. Paraffin 40 min x2 6. Embedding store at 4 degrees.
Aldehydes: Organic compound containing the group.
includes formaldehyde known as formalin and glutaraldheyde which is a colourless
solution in water
The tissue is fixed by cross linkages formed in the protein particularly between lysine residues
These cross linkages do not harm structure of protein strongly so that the antigenicity is not lost.
Lastly formaldehyde is good for immune peroxidase techniques .Which is immonostain used to molecular biology .
Formalin a collarless solution of formaldehyde In water.
Penetrates tissue well but Its really slow
Standard solution is 10%neural buffered formalin
Buffer prevents acidity that would promote autolysis and cause precipitation o formol heme pigment in the tissues.
Glutaraldheyde :a compound used especially in tanning leather and
Causes the deformation of alpha Felix structures in protein and it isn’t good or immunoperoxidee staining .
It fixes quickly and is good for electron microscopy
However penetrates very poorly but gives overall best cytoplasmic and nuclear detail.
Standard solution is a 2% buffered glutaraldyde
Alcohol
Incldudes methyl alcohol and ethyl are protein denatures and are not used for routinely for tissues because they cause too much brittleness and hardness.
Very good for cytological smears because they act quickly and give good nuclear detail .
Spray cans of alcohol fixatives are marketed physicians doining PAP (Test for Cervical Cancer)smears but cheap hairsprays do just well .
oxidizing agents
(is a substance that has the ability to oxidize other substances — in other words to cause them to lose electrons.
Include perm grate fixatives potassium permanganate ,dichromate fixates potassium dichromate and osmium tetroxide.
They Cross link proteins but cause extensive denaturation .
They are specialised applications but are used very infrequently.
Picrate’s
Include fixatives with pirtic acid
It has an unknown mechanism for action
Nuclear detail is good but does not cause as much hardness.
Piratic acid is an explosion hazard in dry form.
As a solution it stains everything it touches yellow including skin .
Steps in preparing a tissue section ?
- Fixation /Processing
Slide preparation begins with fixation of your tissue specimen. This is a crucial step in tissue preparation, and its purpose is to prevent tissue autolysis and putrefaction. For best results, your biological tissue samples should be transferred into fixative immediately after collection.
- Decalcification describes the technique for removing mineral from bone or other calcified tissue so that good-quality paraffin sections can be prepared that will preserve all the essential microscopic elements.
- Dehydration is step, which involves immersing your specimen in increasing concentrations of alcohol to remove the water and formalin from the tissue.
- Embedding
most cases the tissue requires embedding in a medium, which allows thin sections to be cut cleanly; most tissues for routine histology are embedded in wax blocks. This requires that water is removed from the tissue and progressively replaced by wax, which can be solidified later to make a tissue block suitable for sectioning.After clearing, the tissue is infiltrated with embedding agent, usually melted paraffin or celloidin. After infiltration, the embedding agent is made to solidify so that a firm homogenous mass containing the embedded tissue is obtained
Sectioning
Your tissue specimen is now ready to be cut into sections that can be placed on a slide. The microtome can be pre-set to cut at different thicknesses, but most tissues are cut at around 5 µm. Tissue embedded in paraffin may be sliced very thin. For the majority of microscopic work, sections are between 3 and 10 um thick.
- Staining
Finally, the mounted sections are treated with an appropriate histology stain., it is necessary to remove the paraffin solvent or decorating agent, usually by xylol. This step is omitted in the case of a section which has been embedded in celloidin. The section is then passed through descending strengths of alcohol prior to staining. - Mounting
, it is mounted on a clear glass slide, and covered with a thin glass coverslip. The slide and coverslip must be free of optical distortions, to avoid viewing artifacts. A mounting medium is used to adhere the coverslip to the slide.excess dye is removed by washing with water or alcohol depending upon the solvent of the dye and the section is dehydrated through ascending grades of alcohol. - Observation .
Watch to see changes.
Immunofloruence
a technique for determining the location of an antigen (or antibody) in tissues by reaction with an antibody (or antigen) labelled with a fluorescent dye.
How does immunoflorunce
The specific region an antibody recognizes on an antigen is called an epitope.
There have been efforts in epitope mapping since many antibodies can bind the same epitope and levels of binding between antibodies that recognize the same epitope can vary.
Additionally, the binding of the fluorophore to the antibody itself cannot interfere with the immunological specificity of the antibody or the binding capacity of its antigen. Immunofluorescence is a widely used example of immunostaining and is a specific example of immunohistochemistry. This technique primarily makes use of fluorophores to visualise the location of the antibodies.
