Transcript The Process Of Molecular Cytology: Embedding and Sectioning

The Process Of Molecular
Cytology:
Embedding and Sectioning
Natasha Williams
Dr. Katia Manova
Zuckerman Research Building/ MSKCC
Introduction
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The purpose of embedding and sectioning
is to be able to learn how to identify and
master technologies to detect and analyze
molecules in cells, tissues, organs, tumors
and embryos, while being in their natural
environment.
Immunohistochemistry
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Embedding and Sectioning falls under
Immunohistochemistry :the process of
locating antigens in tissue sections by the
use of labeled antibodies as specific
reagents through antigen – antibody
interactions. These interactions are
imaged by a marker ( enzyme ,fluorescent
dye, or radioactive element).
Facts About Immunohistochemistry
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A man named Albert H. Coons and his
colleagues were the first to label antibodies with
a fluorescent dye & use it to identify antigens in
tissues sections.
Many imunohistochemistry methods can be
used to locate antigens.
Has become a major technique and is used in
various types of medical research labs and
clinical diagnostics.
Embedding
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To embed, the embryos, organs, tissues or tumors are
retrieved from the desired animal. Usually a mouse .
Then the tissue or organ, etc is placed into a fixative.
This would allow the tissue to be preserved for a long
period of time.
Formalin Solution ( 10% unbuffered)-----Fixatative Recipe
Formaldehyde (37-40%)------10ml
Distilled Water-------------------90ml
Mix well.
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Then after fixation, the tissue is embedded in paraffin.
Paraffin allows the tissue to be cut into microscopic
sections . Ranging from 4- 10 microns.
Continued..
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With paraffin embedding the main thing is to
dehydrate the tissue so there is no water, and
“clearing”. The tissues are dehydrated with
various alcohols. And “clearing” is the removal of
the dehydrant, may commonly be done with the
agent xylene.
Putting the tissue into paraffin blocks is an
important job because the tissue have to be
aligned properly into the block .
Objective
My objective was to learn how to cut paraffin
sections and mount them onto slides. To be
used in medical research as controls and
experimental slides.
 This was my objective since I was in an area
where researchers were trained to embed and
cut sections. And since I wasn’t to the mastery
level where I could go further with my staining.
Materials
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Ethanol 95% ( flammable liquid)
Slide warmer
Tissue Prep Flotation Bath model 134
Clipboard/ Flat surface to lay ribbons of tissue
Slide dryers- allows water to drain from slides after tissues are mounted.
Latex Gloves
Razors – single edged & for the microtome
Color frost/Plus Microscope slides,precleaned.
Pencil
2 paintbrushes
Leica RM 2155- the model of microtome that was used
Microscope
Kimwipes
U.S Water filter, Deionized water
Process For Tissue Sectioning
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Start off with a clear neat working area.
Get the paraffin block of desire from 4 degree freezer.
Then the water bath have to be filled from the water filter and it has to be
dispensed at 18.2mΩcm.
The water bath has to be turned off . It takes 30 min to prepare.
The paraffin block has to be removed from the container with a razor.
A square block has to be made around the embedded tissue.
The block is then put onto the microtome and adjusted to the cutter’s
comfort.
The size of the tissue sections have to be chosen, 1-10 microns.
The razor is placed into the base of microtome and locked into place.
The cutting can begin and as the ribbon of tissue is forming the paintbrush
is used to guide it.
Then once cutting is done, the water bath should be ready. And 3 or more
tissues can be mounted onto a slide after a few minutes.
Then once mounted, the slide should be placed onto the drying board.
Hematoxylin Eosin
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Known as H&E Staining
Hematoxylin –nucleus—purple
Eosin---cytoplasm--red
Hematoxylin Eosin
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This is one of many stains that can be done with
slides.
An important general stain combination.
Hematoxylin—a natural dye product
Eosin—is an aniline dye.
This stain can be used after any fixation.
References
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http://mskwebs.mskcc.org
http://www.mskcc.org/mskcc/html/62311.cfm
http://www.bu.edu/histology/m/append02.htm
http://www.childrenmrc.org/research_histology/
Other_Histology_Resources/
http://www.ihcworld.com.introduction.htm#ar
Acknowledgements
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Dr. Sat
Susan Vincent
Harlem Children Society
Dr. Katie Manova
Sandra
Mensru
Thank You!!