INFILTRATION PDF Histological Techniques

INFILTRATION / IMPREGNATION - costly - 2-3 changes - permits the entry of a wax/resin to provide a form of support needed du...

INFILTRATION / IMPREGNATION - costly - 2-3 changes - permits the entry of a wax/resin to provide a form of support needed during the microtomy process Vacuum - best method to use - various waxes and polymers are used to replace the clearant found in the Processing - applying pressure / vacuum to tissues tissue - eliminate air pockets (quickest) - requires several changes of an infiltrating agent to displace the clearing - remove bubbles inside the wax agent - allow permeation of infiltrating agents to enter after - solidifies the tissue to facilitate structural rigidity and ease of cutting removing the vacuum PROPERTIES OF INFILTRATING MEDIUM Miscible (can be mixed) with the clearing agent used; non-polar Must NOT be too dense Cheap and reliable ○ paraffin wax (cheapest and most reproducible) Must be compatible with the reagents in the preceding procedures Formulation must support the structure of the tissue Must NOT contribute opacity to the tissue; transparent IMPREGNATING AND EMBEDDING MEDIA performing microtomy without performing impregnation tissue will have sponge Paraffin - simplest, most commonly used infiltrating agent like appearance — cutting will not be smooth - considered as the best — cheapest and reliable tissue juice / plasma: found in between the spaces of tissues — removed when - high reproducibility and reliability performing dehydration - mixtures of purified wax and various additives - ex. plastic resins, antioxidants, dyes, other METHODS OF INFILTRATION additives, and hydrocarbons - solid at room temperature Manual - infiltration by hand - melting point (MP): approx. 60℃, melts faster at Processing - cost-effective (Manual labor of Histotechnician 65 - 70℃ independent of Machines) - density impacts MP (more paraffin, - good for monitoring of the infiltration completeness lower MP) - prone to human errors - most commonly used for routine histopathology has - tedious / time consuming an MP of 56-58℃ - 3-4 changes - recommended temp for setting: 50-60℃ - Tissue sectioning: 4-6 um Automatic - Automated Tissue Processor Processing - performs dehydration, clearing, and infiltration when it hardens it is NOT stiff — has a clay-like or bubble-like - free of / correct / eliminate human errors consistency - monitors temperature and time of agent changing Paraffin can not be used in fatty tissues: Liver — fats need to - constant agitation (faster) be treat off Alternative An alternative to paraffin is necessary when these are observed: Epoxy can be used as an alternative for paraffin — used as Infiltration - processing will remove several components of the shiner or final coating for cars Media tissue that is for investigation - using heat may detrimentally affect several Paraplast - more elastic — easier to mold components of the cell (enzymes, lipids) - difficult to clean - thin sections are necessary - has an unpleasant smell when heated - In electron microscopy, usage of softer wax - mix of highly purified paraffin and synthetic plastic is recommended — possible to break polymers - the infiltrating medium cannot support the tissue - MP: 56-57℃ as it is not that hard - more elastic and resilient than paraffin - advisable for large dense tissue blocks (e.g., bones, Substitutes come in the form of: brain) Resins and other waxes Celloidin (best alternative for paraffin) Embeddol - synthetic wax substitute — similar to Paraplast Agar (in rare cases, for soft tissues) - MP: 56-58℃ ○ agarose agar Gelatin Examples: Plastics Bio/aid – recommended for embedding dyes ○ can be an additive to harden other media Tissue/Mat – paraffin with rubber; same property as that of Paraplast - can come in one or in combination with the other Ester Wax - harder than paraffin Resins - used for embedding and infiltrating tissues for special - has a lower MP vs paraffin — 46-48℃ procedures - soluble with organic solvents (i.e. 95% ethanol, - ex. EM, ultra-thin sectioning for high clearing agents) resolution studies, undecalcified bone, IHC - indicated if cellosolve or xylene is used as a - provides an exceptionally strong support to tissues clearing agent - allows for a thinner sectioning vs. paraffin - may be used as an infiltrating agent if clearing will - recommended for EM be omitted (not done) Examples of currently available resins include epoxy, acrylic, and can be used in EM polyester formulations, either pure or mixtures of these: Advantage: capable of skipping clearing procedure Polyester Resins less dense than water — performing both clearing and at ○ first used among the three (since 1950s) the same time infiltration easily burn and blackens Epoxy Plastics ○ Bisphenol A (Araldite) Water-soluble - Plastic polymers (PEG: Polyethylene glycol) ○ Glycerol (Epon) Waxes - MP: 38-42℃ / 45-56℃ ○ Cyclohexane dioxide (Spurr) - blended with histological paraffin wax to improve Acrylic Plastics adhesion, hardness and plasticity of the agent ○ Glycol methacrylate (GMA) - these are biodegradable plastics ○ Methyl Methacrylate (MMA) Carbowax - PEG based water-soluble wax - generally used as an embedding agent for frozen - may be used if dehydration and clearing will be sections and for delicate specimens omitted - Double embedding: 1st paraffin, 2nd gelatin - suitable for infiltrating tissues for enzyme histochemistry studies since tissues will not be Agar - last option subjected to too much heat - does not provide enough support during sectioning - highly hygroscopic (absorb moisture) - used on small, friable tissue pieces after fixation to provide additional structure (double embedding — Celloidin / - also known as Nitrocellulose wax after infiltration there will be 2 embedding methods) Collodion - purified nitrocellulose - suitable for specimens with: large, hollow cavities (intestines) CONSIDERATIONS DURING IMPREGNATION dense tissues (bones) General Considerations embryos The temperature must be closely monitored soft, delicate tissue sections of mixed ○ every 5 mins temp checking consistency (brain) Several changes of the infiltrating agent is necessary to remove the - comes as a thin, medium, or thick solutions of clearing agent cellulose dissolved in equal parts of ether and alcohol ○ 3 changes of wax usually use — regardless of what infiltrating medium Two usages: Wet and Dry Celloidin The shorter the time of infiltrating the tissues, the better Wet Whatever agent is used in the infiltration process, the same material is ○ thick / viscous and mixed with 70% alcohol used in the embedding process ○ for delicate tissues & special staining A homogenous agent is necessary ○ tissue is immersed, gradual drying ○ allowing crystallization in the structure Dry ○ when diluting wax, it should be clear or same color of wax ○ thin and mixed with 95% alcohol ○ for larger tissues and routine staining Paraffin Wax ○ applied to tissue, drained off, evaporation The temperature set to melt the wax must be at least 2-5℃ above its MP (rule for all wax) Nitrocellulose - another type of celloidin soluble in an ether-alcohol paraffin wax with an MP of 56℃ is routinely used / solution The ambient temperature will dictate what wax with a preset MP will be Low Viscosity - diluted by additional alcohol Nitrocellulose used: - less viscous ○ RT at 20-24℃ = Wax with 54-58℃ MP - allows for a thinner sectioning ○ RT at 15-18℃ = Wax with 50-54℃ MP - Plasticizers (castor oil) may be added to the solution pure to reduce the tendencies of tissue cracking fresh wax must be filtered before use ○ wiremesh: used to filter wax Gelatin - primarily used in the production of whole organ ○ ex. Green’s no. 904 sections with using a mod’d Gough-Wentworth trimmed wax can be reused (2x max) technique ○ to eliminate water in paraffin wax, heat at 100-105℃ - rarely used; dehydration must be avoided  Other Infiltrating Media Schedules for changing varies per infiltrating media MP temperature varies per infiltrating media Follow manufacturer’s recommendation when using media Some infiltrating medium have advantages, disadvantages, and indications for use ○ (ex. gelatin for frozen sections, using celloidin may take days)