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The last lecture we talked about different type of light microscopy

Fluorescent microscopy
*Fluorescent is adye that emit light. So the idea is if we need to study a structure within tissue, I need to study: 1- Whether this structure is present or not 2- see location of this structure within the cell, is associated with plasma membrane or with the mitochondria whatever organelles. Now the principle is we use antigen antibody reaction ,Already we know that antibody they are react with specific antigen and they are available antibody , suppose you study for example actin or myosin or any other protein , so what will you do ? By an antibody that selectively recognizes this molecule and this is what we call the primary antibody, so you put the solution over slide piece. This primary antibody combines with specific molecule. In order to see where is the primary antibody go? Where is it present? Use the secondary antibody, it recognizes the primary antibody and to the secondary antibody I add a tag, this tag usually is adye or color one of these is the fluorescent molecule. Summary:
primary antibody recognize specifically molecule (actins or myosin)

Secondary antibody recognize the primary antibody (detect the location) Add tag to secondary antibody to see clearly

The fluorescent molecule could be excited and then undergo decay, at both situation it emits wavelengths (light) which is longer, and then the differences between these wavelength is detected .

This is the principle 1-We have specimen 2- we have source of light that will pass through two filters and it will allow only blue light to pass and then the second filter will reflect like the specimen and then passes to second filter and third filter. (3 filter through which they will be selection of certain wavelength to pass which represent color)

by using the fluorescent dye ,for example rhodamine , for that you can see the contrast color , you can see the cell in active division ,mitotic spindle (yellowish greenish color), the rest of the cell cytoplasm (blue) , the other component are (red) so you see Avery beautiful contrast color. *This is electron microscopy (used to magnify or explore smaller object)

Light microscopy Magnification = 1000-1500 times Resolution= 0.2Mm

Electron microscopy = 120000times 1nm

How we study the component of cell: cell culture Cell culture: that means we are going to obtain specific lines of tissues (epithelial cell, myosite or fibroblast) that we want to grow in culture, study the living tissue and see the behavior in culture, because the behavior of cell culture will reflect the behavior as if they are in the body. So the purpose of this is here we have the advantage of studying only one type of cell without the interaction with any other of cell (we do know that cell in the tissue interact with each other ) but we need to specifically study on specific type of cells , you can got Apure colon of these cell and this what we grow in the culture and you put it in an incubator ) (and the cell is grow , it is growing outside the body (invitro) or inside the body (invivo). # If we need to study anything about these cell, ex: Fibroblast, you want to see what are affect, ex the synthesis of collagen. So we can apply those factor whether the drug on those cell and watch what happen to synthesis of collagen. When you put these lines of cell in incubator and you grow , the fast line of cell is primary culture after sometimes those cell grow up and they undergo to division and fill up the plate and I can divide to smaller plate this is called secondary culture. We perform this why ?because cells in culture behave as if there in the living , ex fibroblast will eventually secret or synthesis collagen , myosite as will talk about muscle in future they fused together in order to form the muscle fiber , nerve

cell will extend in axon .

This is different macrographs of the different type of the cell are grown in culture. * the 1st one is shows be the fibroblast (spindle shape cell) 2nd is myoblast which fuse together and eventually form the muscle fiber The 3rd one is oligodendrocyte which is support cell in CNs. The 4th one is tobacco to show stain (fluorescence dye) and the histon protein which associated with chromatin, DNA is wrapped around within the cell.

How we study the component cell: Fractionation


As the name implies fractionation: something to break it. We do know that the cell has different organelle; nuclei, DNA, peroxisome, mitochondria. Again, the same principle I want to study only the specific organelle (I want to study mitochondria or only peroxisome ) .there is away to selectively separate these component only and then I do my study ,so here it's to avoid interference with other component that are present in the cell .(this what we call the fractionation ). *So what do I do? 1- You take the sample (fleshy) 2- Homogenize it (make it a soup): there are different ways to homogenize, use physical means a blender (vibration) or osmotic pressure (different concentration of source) * break the membrane of the cell and create a soup and this is what we call (homogenization). 3- take this soup (homogenate) and put it in centrifuge and you spin it at different speed for different period of time. *to describe the speed by (force of gravity). The next figure is shows the homogenization and there are component has different color. *green color (nuclei), red color (mitochondria, lysosome, peroxesom), blue color (microsome and small vesicle). I take the sample and break it (homogenate) ,take the soap and put it in centrifuge : if I spin them at 1000 g (force of gravity =g) , the nuclei will precipitate , and then

if I take the solute above the precipitate and spin it again at 10000g , mitochondria and lysosome will precipitate . And then if I take the solute and spin it again at 100000 g, microsome, ribosome will precipitate. Now, we come to principle; antibody /antigen reaction, and how we use it??? Antibody interacts with specific antigen, (all functions that are perform in our body, and we have protein that is responsible for this function). Every process either protein or group protein eventually there are responsible for that function. Ex: contraction of muscle, there are interaction between actin and myosin. If it happens any change in expression to actin and myosin, it will defect to in muscular contraction. Ex: blood pressure is regulated by many factors, one of these factors we will take it in physiology; there is gas (nitroxide) which is produced by group of enzymes in the body. It will combine with other enzyme to produce cycloGNB which makes relaxation of smooth muscle, where are they present??? In different vessel organ (such as blood vessel), so it will produce vasodilatation and vasoconstriction (nitroxide is regulate the blood pressure).

Another Ex: septic shock (

: (Sever hypotension; sever vaso dilation and then cardiovascular collapse)

so to define it ,we will come back to the protein that secret nitroxide ,and then see what the factors that make to secret nitroxide .so simply we study the expression of that protein that means what is happens (up regulation or down regulation )?? Because it will affect on the function. Antibody makes injection to the specific material in an experimental animals several time , animal will secret antibody against it and you will make collection to the serum , and this antiserum have antigen determination or epitope , you make purification to this serum that contain specific antibody to the material that you injected it in experimental animals .

This is the structure for antibody which has light chain and heavy chain. So simply ,how we go about this? When we want to study expression of protein or a factor, study under experimental condition, I should have compression between what is normal (control) and up normal (experimental). 1st of all I collect samples (control vs. experimental). 2nd homogenization to tissue, we can extract whatever we need; if I need to study the protein we can extract

only protein component, if I need to study DNA we can extract only DNA. *We use different chemical that makes separation to these component that I need it. After we extract all protein, we take these proteins and add it to polyacrylamide which has channel. ( ) , * It have different walls, each wall to specific type of protein (if it control or experimental) When we submit this gel in electric field, the protein will migrate according to the charge and molecular weight, and after migrating the protein will denature (it will become only chain of amino acid )

* Each band represents protein. *we can use appositive sample is 40000. We use also, a control and experiment. *The thickness of band reflects the expression of protein. * the expression in the experimental specimen is up regulated ,this protein is much more up regulated than in the control ,this is explain why the production of the nitroxide increase which makes septic shock .

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The end Best wishes: Ansam alrawashdeh