Histology Lab
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    We will be spending three weeks on this histology project.  The weeks will work out as follows:

Week 1:  Fixation and embedding

Week 2:  Sectioning

Week 3:  Staining

 

Your Histology Handout

            Histology is the study of tissues.  We are going to use histology in our class to study tissues as well as individual cells.  If we tried to view fresh tissue on a light microscope, it would be difficult to get a thin enough sample to view.  It would also be difficult to see any structures clearly without applying stains, and the cells within the tissue would be decomposing throughout our investigation.  Therefore, histology requires learning the process of treating tissues for viewing in the light microscope.  This requires many special techniques and protocols, but fresh tissue treated this way retains much of its composition from within the living organism.  “As soon as a tissue ceases to be alive, its cells start to change.  Multiplying bacteria begin to destroy them, and the process of autolysis (self-digestion) by contained enzymes begins to dissolve them... these cell changes are called postmortem conditions and must be prevented if tissue is to be examined in the laboratory.”[1]

            In addition to preventing postmortem conditions, one needs to maintain cellular structure while increasing resolution of cellular components for light microscopy.  “... it is also necessary to treat tissue to differentiate the solid phase of the protoplasm from the aqueous phase, to change cell parts into materials that will remain insoluble during subsequent treatment, and to protect cells from distortion and shrinkage when subjected to such fluids as alcohol and hot paraffin.”  How is this accomplished?  The process is called fixation, and requires treatment of the tissue with a solution called a fixative.  “Any fixative should:

1.      Penetrate rapidly to prevent postmortem changes.

2.      Coagulate cell contents into insoluble substances.

3.      Protect tissue against shrinkage and distortion during dehydration, embedding, and sectioning.

4.      Allow cell parts to be made selectively and clearly visible.”

Finding a fixative that can perform all of these steps is not necessarily simple.  Different tissues have different penetration requirements and cellular contents.  Also, some fixatives will only allow for certain staining protocols (for cellular contrast enhancement).

            Certainly, the first requirement of fixatives, quick penetration, can be assisted by other techniques.  If one is working on thick tissue, one can either slice it thinner or macerate it to enable quicker fixative action.  Perfusion of fixative (running it through the vasculature of the organism) is also sometimes necessary for proper and rapid tissue fixation; this is carried out on isolated organs by injecting the fixative into a large artery with a syringe.  When fixing organisms with thick exoskeletons, it may also be necessary to make incisions in the exoskeleton to enable faster fixative penetration.

            Some solutions that can work as fixatives are:  acetone, alcohols, and aldehydes.  Many other chemicals are often included in fixatives, to increase their ability to preserve specific cellular components.  For example, picric acid is an excellent protein coagulant and enhances staining of tissue in acid dyes, but it causes a lot of shrinkage and is a very dangerous chemical to use in the lab (if not maintained moist, it can explode upon opening).  Other fixatives may penetrate quickly, preserve and protect the tissue, and allow for staining, but in the process harden the tissue; this makes sectioning of the tissue much more difficult.  We will be using Carnoy's fixative in this course, and that as well as some other fixatives are provided as attachments.

            After fixation, one must usually wash out the fixative.  It cannot be left within the tissue, since its presence typically interferes with future steps in the preparation of the tissue and slides.  This step of washing the tissue can be very important.  Thorough removal of fixative may be important.  Also, if the fixative had a high alcohol percentage, washing in a dilute solution would be a mistake since rapid water entry into the tissue will cause damage.  Typically, one washes the tissue in a series of solutions to fully extract the fixative, taking enough time in each wash to fully replace the tissue’s fluids.  The washes may be in water or in some particular percentage of alcohol.

            If you think about it, you should realize that the thick piece of tissue that you have preserved cannot be stained as is.  In order to stain individual cells, they must be more directly exposed to the stain.  This requires taking thin slices of the tissue, where the thickness of the slices should be approximately the diameter of individual cells.  The tissue cannot be sliced that thinly without embedding it into a denser matrix, because it would fall apart.  The matrix (or media) must actually infiltrate the tissue (rather than just surround it) or the slices will crumble.  Typically, the media is not a water-soluble material; therefore, if the tissue is in an aqueous solution, it must be dehydrated before it can be infiltrated with the embedding media.  Based on these needs, the next steps in preparation of the tissue are dehydration and embedding.

            Dehydration may take a few steps.  One cannot subject their tissue to a change from water to 100% alcohol in one quick change... tissue damage would be extensive.  Therefore, one typically uses a series of gradual changes, moving tissue through:  water, 30% EtOH, 50% EtOH, 70% EtOH, 80% EtOH, 95% EtOH, 100% EtOH.  Other alcohols can be used for dehydration rather than just ethanol (e.g., isopropyl alcohol, butyl alcohols, and methanol), but the standard (and the one that seems to work best) is ethanol; the other alcohols may have subtle negative effects when one reaches the next steps for embedding.  In addition, some tissues are rather sturdy, and depending on the investigator’s needs, one may not have to go through all the intermediate steps.

            Note:  when tissue is transferred to a 100% solution, as soon as it enters that solution (with the previous solution still within the tissue), the integrity of the 100% solution is compromised.  Therefore, any change to a 100% solution will require at least 2 changes:  from solution A to 100% solution B, and then into 100% solution B again.

            We will be embedding in paraffin in our class.  Other embedding media are available (e.g., epoxy-types of resins), but could require a different set of instruments for sectioning.  Paraffin is a liquid when heated to approximately 55-60°C.  Note that this temperature can cause problems for some tissues, but that typically fixation of tissues allows them to withstand this high temperature.  The tissue as we have described it will be in 100% ethanol at this point, but paraffin is not soluble in ethanol (or any of the other alcohols mentioned).  It is, however, soluble in xylene and in toluene.  Therefore, the tissue will have to be transferred into xylene before embedding can begin.  This step is also called clearing the tissue, since immersion in xylene causes the tissue to become somewhat transparent.  (note:  Xylene also hardens the tissue, making it a bit brittle).

            The steps for embedding in paraffin include the following solution changes:  100% xylene, 100% xylene, 1:1 100% xylene : paraffin, pure paraffin, pure paraffin.  Once paraffin is included in the solution changes, the tissue and solution must remain inside a paraffin oven so that it remains a liquid.  Also, all of the initial solution changes should take place in glass containers, since xylene dissolves plastic.  Once the tissue is finished in the second pure paraffin solution, it is ready for its final step for embedding.

            Now the tissue can be transferred to an embedding chamber that contains pure paraffin.  It is in this chamber that the paraffin will be allowed to harden, now containing the tissue, for sectioning.  However, if the tissue (which is totally transparent) is just dropped into the chamber and then the paraffin is allowed to harden, it will be impossible to find the tissue within the paraffin block for proper sectioning.  Therefore, the chamber must be kept warm (by placing it on a warming plate) so that the tissue can be properly oriented.  After dropping the tissue into the chamber, warm forceps can be used to manipulate the tissue into a specific location and orientation within the chamber.  The chamber can then be marked so that the tissue will be easy to locate after the paraffin cools.

            At this point, we will simply be letting our paraffin chambers cool.  However, if we had an oven with a vacuum pump, we would return the chamber to the oven, apply the vacuum (to suck out any air bubbles in our chamber), double-check the tissue orientation, and then let it cool.

            Once embedded, the tissue must then be sectioned on a microtome.  When sectioning is complete (that will be explained in great detail in class), your tissue sections will now be on a slide.  We have some sample slides with paraffin sections on them.  The sections will store for years in this condition.  The next thing that has to be done before you can stain these sections is you have to deparaffinize the sections.  If you were to look at the slides with all the paraffin on them, you would never see the tissue for all the paraffin that is there.  So the paraffin has to be removed by immersion in xylene or toluene.

            Next, is the staining of the tissue sections.  You will learn about a variety of stains in the weeks to come.  Keep in mind that if the stains are aqueous, you will first have to go back through an alcohol series to rehydrate your tissue before staining can occur.  Finally, after staining is complete, it is time to coverslip your slides for viewing.  If this is for long-term storage, you will typically have to dehydrate your stained tissue before applying the coverslip.  Therefore, staining and coverslipping may take quite some time as you progress back and forth through a number of intermediate steps.

            Now the tissue can be viewed on a microscope!  What do you see?


 

Some Fixatives:

 

Carnoy Fixative A good but tough fixative.  Chloroform is said to make the action rapid, but needs to be fully washed out.  It is considered an important fixative for glycogen and Nissl substance, but it dissolves most other cytoplasmic elements.  Although Humason recommends 3-6 hours of fixation, small pieces of tissue can be fully fixed in 20-30 minutes.

glacial acetic acid................................................................................. 10 mL

100% ethanol...................................................................................... 60 mL

chloroform........................................................................................... 30 mL

 

Buffered FormalinTissue may remain in fixative indefinitely; action of fixative is progressive.  Wash in water.

10% formalin....................................................................................... 1000 mL

sodium acid phosphate (NaH2PO4 · H2O)............................................     4.0 g

anhydrous disodium phosphate (Na2HPO4)..........................................     6.5 g

 

4% Paraformaldehyde Fixative:

Paraformaldehyde....................................................................   4 g

dH2O...................................................................................... 50 mL (total volume)

     Heat solution while stirring to 60°C.  When dissolved and cloudy, add 0.1N NaOH drop-by-drop until clear.

cold 2X buffer......................................................................... to make total volume 100mL

 

Week 1:  Fixation and Embedding Steps:

1.      Cut your tissue down to an appropriate size.

2.      Immerse your tissue in Carnoy fixative for 20 minutes to an hour, depending on the tissue.

3.      Rinse your tissue through the following steps (each for approximately 10 - 20 minutes):

·        70% EtOH

·        (70% EtOH again if necessary)

·        95% EtOH

·        100% EtOH

·        100% EtOH (closed container if possible)

·        100% xylene (xylene will dissolve plastic)

·        100% xylene

--- these next steps must be carried out in an incubator ---

·        1:1 100% xylene : paraffin

·        pure paraffin

·        pure paraffin (see next step)

4.      For this second round in paraffin, the tissue should be placed into the appropriate mold for hardening.  The paraffin and tissue in the mold may then be exposed to a vacuum environment to draw out any air from the tissue or surrounding embedding media.

5.      The tissue must now be quickly positioned within its mold and allowed to harden.


 

[1] All quotes and most recipes have been taken from:  Humason, GL (1979) Animal Tissue Techniques, 4th edition.  WH Freeman and Co., San Francisco.  While there are many sources for histological techniques, this one has proven the best (by far) in my experience.  All other information within this lab handout is taken from the personal experience of your instructor.

 

    As you write your lab report, you might find it easiest to start with the "results" section.  Just write down what you saw.  Then write the "discussion" section, where you explain why you think things looked the way they did and your interpretations of the results.  Don't forget that I'd like your impressions on:

bullethow well each step of our procedure went (meaning, how good was fixation?  How good was the sectioning?  How good was the staining?  why and why not?)
bulletyour impressions of the tissue you selected for the day
bulletyour impressions of how the stain you were responsible for worked on the slides.

    After the results and discussion are written, you might then feel comfortable writing the introduction and methods.  Save the abstract for last, since it is a short (one or two paragraph) summary of the entire lab report.  OK?