Histological Lecture 1 PDF

 ‫قســم الهستولوجيا‬ ‫جامعة جنوب الوادي‬ Histotechinque Presented by Fatma Ahmed Abdelhamid lecturer of Histology 10/1/2022 By the end of lecture, you should have answer for the following What is the meaning of histology? Diffe...

 ‫قســم الهستولوجيا‬ ‫جامعة جنوب الوادي‬ Histotechinque Presented by Fatma Ahmed Abdelhamid lecturer of Histology 10/1/2022 By the end of lecture, you should have answer for the following What is the meaning of histology? Differentiated between the different histological technique? What is aims of using the histological techniques? What is the component of light microscopy?  Introduction Cell: smallest unit of structure and function of the body Tissue: groups of the cells and extracellular ground substance Organ: consisted of tissue and have special shape, structure and function System: made up organs that have related function together  Histological procedures (Making histological sections for the light microscope) For light microscopy, three techniques can be used: the paraffin technique, frozen sections, and semithin sections. The paraffin technique is the most commonly used. Once the sections are prepared, they are usually stained, to help distinguish the components of the tissue.  Histological procedures for paraffin section Tissue Tissue Embedding Tissue Slides Dehydration clearing sampling fixation sectioning staining Histological procedures  Tissue Sampling Tissue sampling should be taken from healthy animal or bird. Washing the tissue with PBS.  Tissue Fixation The process of tissue fixation is aimed to keep the constituents of the cells and tissue fixed with minimum loss of architectures and this happened by using different reagents called fixatives. The fixative prevent the cell autolysis cellular enzymes and bacterial decomposition. Many kind of fixative: Formalin Glutaraldehyde Bouin’s solution  Dehydration & clearing Tissue is place in different concentration of ethyl alcohol: 70% ethanol ,80%,90%,100%(critical point). The ethanol penetrate the tissue and replace the water with alcohol. The clearing process is used to replace the alcohol with the clearing substance as the paraffin wax is not alcohol soluble. There are different clearing solutions: 1. xylene 2. methyl benzoate 3. Benzene  Impregnation in paraffin wax The paraffin wax is melted at 50-60c Place the tissue in 3 different jar in melted paraffin. The duration of paraffin wax infiltration depend on the type of the tissue Embedding in paraffin wax The melted paraffin wax pour on the tissue in a mold. Leaving the paraffin wax to solidified Sectioning by using the microtome  Staining Staining is meaning giving color to the sections The staining steps: 1. Deparaffinization 2. Rehydration 3. Washing 4. Staining 5. Dehydration 6. Mounting and covering  Staining with H&E Hematoxylin is basic stain (nucleus stained basophilic) Eosin is acidic stain (cytoplasm stained acidophilic ) There special stains used for staining a specific components inside the cells like glycogen, Golgi apparatus, elastic fibers H&E Staining Kit (Hematoxylin and Eosin) (ab245880) | Abcam H&E stain - Wikipedia  Histological procedures for the electron microscope(TEM) 1- Tissue Sampling 2- Tissue fixation: used the fixative for prevention the cell autolysis and bacterial decomposition. Glutaraldehyde and osmium tetroxide is used for TEM technique 3- Dehydration : used for with drawing the water from the cells by using different concentration of acetone/ ethanol 4- Embedding in Epoxy Resin 5- Sectioning by using ultra tome by using glass knife 6- Staining by using uranyl acetate and lead citrate less than 1 micron There are thick ultra section (1 micron) stained by toluidine blue  Cryopreservation technique There are many methods for storage: Cryopreservation is meaning storage in liquid nitrogen Cold storage is meaning storage in low or nonfreezing temperature Pressure storage involved partially reducing the atmospheric pressure of the surrounding  Cryo is meaning frost The principle is keeping the tissue in zero metabolism and non dividing state by reducing the temperature in presence of cryoprotectant. It can be done 1. Over solid carbon dioxide -79 2. Freezer -80 3. In vapor phase of nitrogen -150 4. In liquid nitrogen -196 Sucrose solution as cryoprotectant  The embedding media is OCT (optimum cutting temperature) Cut sections 5-15 μm thick in the cryostat at −20°C Staining is labeled antibody used for immunofluorescence and immunohistochemistry staining Green immunofluorescent stain for Nissl's granules  Light microscopy light microscopes are used to see details and enlarged images of small objects. Enlarging an image is called magnification. The amount of fine detail that can be seen is called resolution. Light is focused onto the specimen (i.e. the histology slide) by a condenser. The image produced is magnified by a combination of the objective lens and the eyepiece lens.  Usually, the eyepiece lens gives a x 10 magnification. Three objective lenses are usually used: x10, x40 and x100. The x100 lens is usually an oil- immersion lens - you need to view the sample through a drop of oil. It is difficult to see much detail in live cells under the microscope, because cells tend to be colorless and transparent. This is why cells are often fixed and stained for microscopy.  Good luck Happy Dreams!!!