Let's break down the fixatives and buffers used in electron microscopy. Fixatives are chemicals used to preserve tissue structure as close to its living state as possible by cross-linking proteins and lipids. 1. Glutaraldehyde: Function: It's a dialdehyde that forms stable cross-links between amino groups of proteins, effectively stabilizing cellular components. It's often used as the primary fixative. Formation/Preparation: Typically purchased as a concentrated aqueous solution (e.g., 25% or 50%). It's then diluted to the working concentration (e.g., 2.5% to 4%) using a suitable buffer. It should be stored cold and checked for purity (e.g., by spectrophotometry) as it can polymerize over time, reducing its effectiveness. 2. Osmium Tetroxide (OsO₄): Function: Used as a secondary fixative after glutaraldehyde. It fixes and stains lipids, particularly phospholipids, making membranes visible. It also further cross-links proteins and acts as an electron stain due to its heavy metal nature. Formation/Preparation: Usually supplied as a crystalline solid in sealed ampoules due to its volatility and toxicity. It's dissolved in distilled water or a buffer to a working concentration (e.g., 1% to 2%). It must be handled in a fume hood due to its hazardous vapor. Buffers are solutions that resist changes in pH, ensuring the fixatives act at an optimal pH range to prevent cellular damage or artifacts. 1. Phosphate Buffer (Sorensen's Phosphate Buffer): Function: A widely used buffer for electron microscopy, typically prepared to physiological pH (around 7.2-7.4). It's generally well-tolerated by tissues. Formation/Preparation: Prepared by mixing solutions of monobasic sodium phosphate (NaH₂PO₄) and dibasic sodium phosphate (Na₂HPO₄) in specific ratios to achieve the desired pH. Sometimes, potassium phosphate salts are used. The osmolarity is adjusted with sucrose or salts to match tissue osmolarity. 2. Cacodylate Buffer (Sodium Cacodylate): Function: Another common buffer, particularly useful when phosphate precipitation is a concern (e.g., with calcium-rich tissues). It also buffers effectively at physiological pH. Formation/Preparation: Prepared from sodium cacodylate trihydrate (Na(CH₃)₂AsO₂·3H₂O). The pH is adjusted using hydrochloric acid (HCl). It contains arsenic, so proper disposal is crucial. Osmolarity is adjusted similarly to phosphate buffer. 3. HEPES Buffer (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid): Function: A zwitterionic organic chemical buffer that is effective in the physiological pH range (6.8-8.2). It's often used for live cell imaging or when CO₂ buffering is needed, as it doesn't react with CO₂. Formation/Preparation: Prepared by dissolving HEPES powder in distilled water and adjusting the pH with sodium hydroxide (NaOH) to the desired range (e.g., 7.2-7.4). That's 2 down. 3 left today — send the next one.