1. The Process Of Molecular Cytology: Embedding and Sectioning Natasha Williams Dr. Katia Manova Zuckerman Research Building/ MSKCC

2. Introduction 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. 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.

3. Immunohistochemistry 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). 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).

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

5. Embedding To embed, the embryos, organs, tissues or tumors are retrieved from the desired animal. Usually a mouse. 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. 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. Then after fixation, the tissue is embedded in paraffin. Paraffin allows the tissue to be cut into microscopic sections. Ranging from 4- 10 microns. Then after fixation, the tissue is embedded in paraffin. Paraffin allows the tissue to be cut into microscopic sections. Ranging from 4- 10 microns.

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

7. 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. 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.

8. Materials Ethanol 95% ( flammable liquid) Ethanol 95% ( flammable liquid) Slide warmer Slide warmer Tissue Prep Flotation Bath model 134 Tissue Prep Flotation Bath model 134 Clipboard/ Flat surface to lay ribbons of tissue Clipboard/ Flat surface to lay ribbons of tissue Slide dryers- allows water to drain from slides after tissues are mounted. Slide dryers- allows water to drain from slides after tissues are mounted. Latex Gloves Latex Gloves Razors – single edged & for the microtome Razors – single edged & for the microtome Color frost/Plus Microscope slides,precleaned. Color frost/Plus Microscope slides,precleaned. Pencil Pencil 2 paintbrushes 2 paintbrushes Leica RM 2155- the model of microtome that was used Leica RM 2155- the model of microtome that was used Microscope Microscope Kimwipes Kimwipes U.S Water filter, Deionized water U.S Water filter, Deionized water

9. Process 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 be dispensed at 18.2mΩcm. 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 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 comfort. 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 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 paintbrush is used to guide it. 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 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. Then once mounted, the slide should be placed onto the drying board.

10. Hematoxylin Eosin Known as H&E Staining Known as H&E Staining Hematoxylin –nucleus—purple Hematoxylin –nucleus—purple Eosin---cytoplasm--red Eosin---cytoplasm--red

11. Hematoxylin Eosin This is one of many stains that can be done with slides. This is one of many stains that can be done with slides. An important general stain combination. An important general stain combination. Hematoxylin—a natural dye product Hematoxylin—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.

12. References http://mskwebs.mskcc.org http://mskwebs.mskcc.org http://mskwebs.mskcc.org http://www.mskcc.org/mskcc/html/62311.cfm http://www.mskcc.org/mskcc/html/62311.cfm http://www.mskcc.org/mskcc/html/62311.cfm http://www.bu.edu/histology/m/append02.htm http://www.bu.edu/histology/m/append02.htm http://www.bu.edu/histology/m/append02.htm http://www.childrenmrc.org/research_histology/ Other_Histology_Resources/ http://www.childrenmrc.org/research_histology/ Other_Histology_Resources/ http://www.childrenmrc.org/research_histology/ Other_Histology_Resources/ http://www.childrenmrc.org/research_histology/ Other_Histology_Resources/ http://www.ihcworld.com.introduction.htm#ar http://www.ihcworld.com.introduction.htm#ar

13. Acknowledgements Dr. Sat Dr. Sat Susan Vincent Susan Vincent Harlem Children Society Harlem Children Society Dr. Katie Manova Dr. Katie Manova Sandra Sandra Mensru Mensru Thank You!!