Histology Lecture 1 Microtechniques PDF

# Histology - Lecture 1 ## Introduction - Micro technique - Paraffin technique: - Can be made easily but melts easily so it causes tissue damage. - Frozen technique: - Extra note - Cellordine technique: - A method of hardening by adding Cellordine and it gi...

# Histology - Lecture 1 ## Introduction - Micro technique - Paraffin technique: - Can be made easily but melts easily so it causes tissue damage. - Frozen technique: - Extra note - Cellordine technique: - A method of hardening by adding Cellordine and it gives support to the tissue. ## Paraffin Technique ### Characteristics of the Sample 1. Take it immediately in order not to spoil. 2. Cut it into small pieces before putting it into paraffin. ### Steps of paraffin technique 1. Taking tissue sample 2. Fixation - 10% Formaldehyde - The aim of formaldehyde? - Preserves the fine structure of tissues - Prevents the autolysis by inhibition of hydrolytic enzymes of lysosomes - Prevents putrefactions by killing bacteria. - Hardens tissue due to coagulation of proteins. - Increases the affinity of tissues to staining effects. 3. Dehydration - Removing of water from the sample to stay longer without spoiling from anything as bacteria/fungus - Dehydrating agent: Alcohol - Procedures: Boiling Alcohol in ascending degree to prevent tissues' shrinkage: So, water is replaced and Alcohol with organic solvent. 4. Clearing - Putting the sample in paraffin solvent to react with paraffin and alcohol. - Ben Zene derivative: Xylol. - We don't use benzene directly as it is sharp on the tissue and may burn it, so we use one of its derivatives. 5. Impregnation - Melted soft paraffin to penetrate inter cellular spaces 6. Embedding - Melted hard paraffin to make it a hard block 7. Sectioning - Cutting it into 5-8 µm sections by microtome. 8. Mounting - Putting the sample on glass slide for easy handling. ## Different Staining Techniques +Ve -Ve 1. Basic & Acidic Stains - Hematoxylin & Eosin Stain (H&E) - Basic Acid - Acidic = Base | | | |:-:|---------------------------------------------------------------| | **philic** | **like** | | Basophilic | like basic stains - Nucleus DNA, RNA, Ribosomes | | Acidophilic | like acidic stains - Cytoplasm, muscle | - Basic Stains: Hematoxylin & toluidine blue - Acidic Stains: Eosin & Orange G 2. Neutral Stains - Base + Acid - The tissue may show affinity for the acid dyes, basic dye, or for the whole compound. - exe-Leishman's stain in blood film 3. Vital stain - Staining of living cells inside the living body. - Stain-trypad blue - Methods -Injecting the dye in an animal, then the Phagocytic Cells 'll engulf the dye, as any foreign body so the cells'll be stained. 4. Supravital stain - Staining of living cells outside the body - as Mitochondria with Janus green B Stain 5. Metachromatic stain - Staining of a certain cell component with a color different from that of original dye - Mast cell stained with Toluidine blue 'll appear red in color (Metachromatic reaction). Due to containing high percentage of protein. So it reflects the stain with different color. 6. Histochemical stain - Identify chemical structures of the cell - Enzymes - Carbohydrates - Lipids - exe - Staining the glycogen (carbohydrates) in liver cell by PAS stain. - Staining fat by Sudan III. - **Note:** By using H&E Stain carbohydrtes'll not appear as the Carbohydrates'll be dissolve due heating at 50°/60° and the lipids too. 7. Immunohistochemistry - Identifying and localize specific proteins as antigen and antibody 8. Special stains - Staining "Golgi apparatus" with silver stain - brown & colour. ## Microscopes | | | | :-------------- | :--------------------------------------- | | Light microscope | - Beam of light | | | - Colourful | | | - No details inside the cells | | Electron microscope | - White & Black | | | - Bars show details | - Transmission - 2d. - TEM - Scanning - 3d. - SEM