The Process Of Molecular The Process Of Molecular Cytology: Cytology:

Embedding and SectioningEmbedding and Sectioning

Natasha WilliamsNatasha Williams

Dr. Katia ManovaDr. Katia Manova

Zuckerman Research Building/ Zuckerman Research Building/ MSKCCMSKCC

IntroductionIntroduction

The purpose of embedding and The purpose of embedding and sectioning is to be able to learn how sectioning is to be able to learn how to identify and master technologies to identify and master technologies to detect and analyze molecules in to detect and analyze molecules in cells, tissues, organs, tumors and cells, tissues, organs, tumors and embryos, while being in their natural embryos, while being in their natural environment.environment.

ImmunohistochemistryImmunohistochemistry

Embedding and Sectioning falls under Embedding and Sectioning falls under ImmunohistochemistryImmunohistochemistry :the process of :the process of locating antigens in tissue sections by locating antigens in tissue sections by the use of labeled antibodies as specific the use of labeled antibodies as specific reagents through antigen – antibody reagents through antigen – antibody interactions. These interactions are interactions. These interactions are imaged by a marker imaged by a marker ( enzyme ,fluorescent dye, or radioactive ( enzyme ,fluorescent dye, or radioactive element). element).

Facts About Facts About ImmunohistochemistryImmunohistochemistry

A man named Albert H. Coons and his A man named Albert H. Coons and his colleagues were the first to label colleagues were the first to label antibodies with a fluorescent dye & use it antibodies with a fluorescent dye & use it to identify antigens in tissues sections.to identify antigens in tissues sections.

Many imunohistochemistry methods can Many imunohistochemistry methods can be used to locate antigens.be used to locate antigens.

Has become a major technique and is Has become a major technique and is used in various types of medical research used in various types of medical research labs and clinical diagnostics.labs and clinical diagnostics.

EmbeddingEmbedding

To embed, the embryos, organs, tissues or tumors To embed, the embryos, organs, tissues or tumors are retrieved from the desired animal. Usually a are retrieved from the desired animal. Usually a mouse .mouse .

Then the tissue or organ, etc is placed into a Then the tissue or organ, etc is placed into a fixative. This would allow the tissue to be preserved fixative. This would allow the tissue to be preserved for a long period of time.for a long period of time.

Formalin Solution ( 10% unbuffered)-----Fixatative Formalin Solution ( 10% unbuffered)-----Fixatative RecipeRecipeFormaldehyde (37-40%)------10mlFormaldehyde (37-40%)------10mlDistilled Water-------------------90mlDistilled Water-------------------90mlMix well.Mix well.

Then after fixation, the tissue is embedded in Then after fixation, the tissue is embedded in paraffin. Paraffin allows the tissue to be cut into paraffin. Paraffin allows the tissue to be cut into microscopic sections . Ranging from 4- 10 microns.microscopic sections . Ranging from 4- 10 microns.

Continued..Continued..

With paraffin embedding the main thing is to With paraffin embedding the main thing is to dehydrate the tissue so there is no water, dehydrate the tissue so there is no water, and “clearing”. The tissues are dehydrated and “clearing”. The tissues are dehydrated with various alcohols. And “clearing” is the with various alcohols. And “clearing” is the removal of the dehydrant, may commonly removal of the dehydrant, may commonly be done with the agent xylene.be done with the agent xylene.

Putting the tissue into paraffin blocks is an Putting the tissue into paraffin blocks is an important job because the tissue have to be important job because the tissue have to be aligned properly into the block .aligned properly into the block .

ObjectiveObjective

My objective was to learn how to cut paraffin My objective was to learn how to cut paraffin sections and mount them onto slides. To sections and mount them onto slides. To be used in medical research as controls be used in medical research as controls and experimental slides.and experimental slides.

This was my objective since I was in an This was my objective since I was in an area where researchers were trained to area where researchers were trained to embed and cut sections. And since I embed and cut sections. And since I wasn’t to the mastery level where I could wasn’t to the mastery level where I could go further with my staining. go further with my staining.

Materials Materials

Ethanol 95% ( flammable liquid)Ethanol 95% ( flammable liquid) Slide warmerSlide warmer Tissue Prep Flotation Bath model 134Tissue Prep Flotation Bath model 134 Clipboard/ Flat surface to lay ribbons of tissueClipboard/ Flat surface to lay ribbons of tissue Slide dryers- allows water to drain from slides after tissues are Slide dryers- allows water to drain from slides after tissues are

mounted.mounted. Latex GlovesLatex Gloves Razors – single edged & for the microtomeRazors – single edged & for the microtome Color frost/Plus Microscope slides,precleaned.Color frost/Plus Microscope slides,precleaned. PencilPencil 2 paintbrushes2 paintbrushes Leica RM 2155- the model of microtome that was usedLeica RM 2155- the model of microtome that was used MicroscopeMicroscope KimwipesKimwipes U.S Water filter, Deionized water U.S Water filter, Deionized water

Process For Tissue SectioningProcess For Tissue Sectioning

Start off with a clear neat working area.Start off with a clear neat working area. Get the paraffin block of desire from 4 degree freezer.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 Then the water bath have to be filled from the water filter and it has to

be dispensed at 18.2mbe dispensed at 18.2mΩΩcm.cm. The water bath has to be turned off . It takes 30 min to prepare.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.The paraffin block has to be removed from the container with a razor. A square block has to be made around the embedded tissue.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 The block is then put onto the microtome and adjusted to the cutter’s

comfort.comfort. The size of the tissue sections have to be chosen, 1-10 microns.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 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 The cutting can begin and as the ribbon of tissue is forming the

paintbrush is used to guide it.paintbrush is used to guide it. Then once cutting is done, the water bath should be ready. And 3 or 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.more tissues can be mounted onto a slide after a few minutes. Then once mounted, the slide should be placed onto the drying board.Then once mounted, the slide should be placed onto the drying board.

Hematoxylin EosinHematoxylin Eosin

Known as H&E StainingKnown as H&E Staining Hematoxylin –nucleus—purpleHematoxylin –nucleus—purple Eosin---cytoplasm--redEosin---cytoplasm--red

Hematoxylin EosinHematoxylin Eosin

This is one of many stains that can be This is one of many stains that can be done with slides.done with slides.

An important general stain combination.An important general stain combination. Hematoxylin—a natural dye productHematoxylin—a natural dye product Eosin—is an aniline dye.Eosin—is an aniline dye. This stain can be used after any fixation.This stain can be used after any fixation.

ReferencesReferences

http://mskwebs.mskcc.orghttp://mskwebs.mskcc.org http://www.mskcc.org/mskcc/html/62311.chttp://www.mskcc.org/mskcc/html/62311.c

fmfm http://www.bu.edu/histology/m/append02.http://www.bu.edu/histology/m/append02.

htmhtm http://www.childrenmrc.org/research_histohttp://www.childrenmrc.org/research_histo

logy/Other_Histology_Resources/logy/Other_Histology_Resources/ http://http://

www.ihcworld.com.introduction.htm#arwww.ihcworld.com.introduction.htm#ar

AcknowledgementsAcknowledgements

Dr. SatDr. Sat Susan Vincent Susan Vincent Harlem Children Society Harlem Children Society Dr. Katie ManovaDr. Katie Manova SandraSandra MensruMensru

Thank You!!Thank You!!