Histopathology | Microtomy to Deparaffinization MTAP 2nd Sem PDF

HISTOPATHOLOGY | MICROTOMY TO DEPARAFFINIZATION MTAP 2ND SEM | A.Y. 2024-2025 ​ TISSUE BLOCK - the product of embedding, ​ Standard sliding — to be used in cutting. And cutting tissues in to MOVABLE P...

HISTOPATHOLOGY | MICROTOMY TO DEPARAFFINIZATION MTAP 2ND SEM | A.Y. 2024-2025 ​ TISSUE BLOCK - the product of embedding, ​ Standard sliding — to be used in cutting. And cutting tissues in to MOVABLE PART (Knife); thin slices is what we call SECTIONING. STATIONARY (Block Holder); ​ SECTIONING — involves cutting tissue into requires a slow but very thin slices. And in order to cut tissue sections, steady motion during microtomes are used. manipulation ​ Freezing microtome — Invented by T TYPES OF MICROTOME QUECKETTE; uses (1) intermittent burst of ​ Ultrathin Microtome — intended for carbon dioxide (freezing agent) to freeze the M ELECTRON MICROSCOPY; thickness of block holder and the tissue (2) it has cooling sections produced is 0.5 micra; tissues are device that can lower the knife temp; usually embedded in plastic Thickness of sections 10-15 u; can ,R ​ Rotary microtome — was developed by immediately harden tissues without prior MINOT and it is used to cut paraffin fixation embedded tissue; the MOST COMMON and ○​ PURPOSE: commonly used for research purposes; ​ Demonstration of fats and thickness of sections 4-6 microns other neurological ES ​ Rocking microtome — invented by structures TREFALL; thickness of sections 10-12 micra; ​ Used to cut tissues with heat used to prepare serial sections of large sensitive structures. paraffin block; MOST SIMPLE ○​ FREEZING AGENTS: ○​ DISADVANTAGES: ​ Liquid nitrogen EL ​ Difficulty in re-orienting the ​ Isopentane cooled by liquid block nitrogen ​ Restriction in the size of block ​ Dry ice that can be cut ​ Carbon dioxide gas ​ Aerosol sprays G ​ Sliding microtome — invented by ADAMS; thickness of the section 7-9 micra; MOST ​ Cryostat / Cold microtome — the DANGEROUS because of the exposed knife temperature inside the chamber will AN ○​ PURPOSE: immediately harden tissues to facilitate ​ For celloidin embedded immediate cutting; For fresh tissue microtomy, tissue or ester infiltrated refrigerated (-5 to -30 degC) average is -18 to tissue -20 degC; uses cryostat – a refrigerated ​ To cut extremely hard or tough apparatus (a rotary microtome) enclosed in a tissues cold chamber ○​ TYPES: ○​ PURPOSE: ​ Base sledge — MOVABLE ​ For preparing thin sections of PART (Block Holder); fresh frozen tissues for STATIONARY (Knife); less fluorescent antibody dangerous 1  staining or histochemical concave (paraffin embedded); flat enzyme studies celloidin embedded) ​ Freezing microtome uses freezing agent to ○​ Plane wedge – for frozen sections; immediately harden the blocks, while cryostat extremely hard or tough tissues; uses the temp inside the chamber to harden both sides are flat the blocks ○​ Disposable blades – commonly used today; size of sections 2-4 u FROZEN SECTIONS ○​ Diamond knife and glass knives – ​ In the laboratory, we usually prepare 2 types for electron microscopy of sections: ​ Diamond knives — are brittle T ○​ Paraffin section — we use ROTARY and quite expensive; cuts MICROTOME resin blocks for EM M ○​ Frozen section – type of tissue ​ Glass knives — used for section usually requested for RAPID trimming and semi-thin DIAGNOSIS; we prepare frozen sectioning of tissue blocks for ,R section to come up with diagnosis electron microscopy while the patient is in the OPERATING ROOM processing fresh specimen HONING & STROPPING ​ 2 METHODS OF PREPARING FROZEN ​ Honing — process of grinding the cutting SECTIONS: edge to have an even edge ES ○​ Cold knife procedure – requires ○​ Purpose: To remove gross nicks freezing microtome; use of freezing ○​ Material for honing: Oil stones and agent to immediately harden fresh hones tissues for immediate cutting; tissue ​ Belgium yellow — type that blocks should be 3-5 mm; obsolete gives the best result EL ○​ Cold microtome / Cryostat method – ​ Arkansas — gives a more uses cryostat; preferred nowadays polishing effect than belgium ​ Cryostat and freezing microtome are used for yellow FRESH TISSUES ​ Fine carborundum — G recommended for badly MICROTOME BLADES nicked knives; with a much ​ Honing and stropping are not done if we coarser bite AN utilized disposable blades ○​ Procedure: Heel to toe movement, ​ To cut tissues TRADITIONALLY, we use edge first microtome knives, but now we commonly use ○​ Number of strokes required: 20-30 DISPOSABLE BLADES strokes ​ MICROTOME BLADES: ○​ During honing, we need to use ○​ Biconcave – for cutting paraffin lubricants like soapy water, xylene, embedded tissues; both sides of the etc. knives are concave ​ Stropping — process of polishing and ○​ Plane concave – one side of the knife sharpening cutting edge is concave, while the other one is flat; ○​ Purpose: to remove burrs and other irregularities formed during honing 2  ○​ Material: Leather strop or horse float out bath in order to remove the wrinkles leather and fold. ○​ Procedure: Toe to heel movement, ○​ The main purpose of float out bath is edge last to FLATTEN THE RIBBONS ○​ Number of strokes: 40-120 double ​ Temperature of the float out bath: 5-10 deg strokes lower than the melting point of the wax, ○​ Do not require lubricant specifically 45-50 deg ○​ Must be OILED prior to use ​ Fishing out — process of removing ribbons ○​ Commonly used oils: Vegetable oils from float out bath like castor oil ​ Orientation — process of placing the tissue T ​ MINERAL OIL should not be in a precise position (middle of the slide) used because it tends to M blister or destroy the leather. ADHESIVES ​ Stropping may be carried out without prior ​ Before fishing out, a slide must be prepared honing and it should be coated with adhesives ,R ​ Stropping must be done after the process of ​ Process: Placing adhesives onto the slides > honing fishing out > orienting ​ Disposable blades will not require honing and ​ Adhesives — promotes adhesion of the tissue stropping; Microtome knives will require honing sections into the slides; to promote attachment; and stropping to prevent detachment of ribbons from slide ES ○​ MAYER’S EGG ALBUMIN — most IMPORTANT ANGLES commonly used adhesive; a ​ Bevel angle — set at 27-37 deg; the angle combination of egg white and formed between cutting edges of the knife glycerine ​ Wedge angle — set at 14-15 deg; the angle ​ equal amount of egg white EL formed by the sides of the wedge knife and glycerine PLUS thymol ​ Clearance angle / tilt angle / inclination crystals in order to prevent angle — is the most important angle the growth of molds because in sectioning we need to follow the ○​ Poly-l-lysine — widely used as a G correct clearance angle to prevent uneven section adhesive in sections; the angle betwen the cutting edge of immunohistochemistry; its the knife and the surface of the tissue effectiveness as an adhesive slowly AN block; set at 0-15 deg, but during sectioning decreases as the time goes by we set it at 5-10 deg to prevent uneven ○​ Apes — very useful for cytology; sections/sections that are alternately thin or recommended for specimen that is thick bloody and has high protein content ○​ Starch paste RIBBONING, FISHING OUT & ORIENTING ○​ Dried albumin ​ Product of sectioning: Thin slices/tissue sections/ribbons DEPARAFFINIZATION ​ The ribbons tend to be wrinkled after ​ Deparaffinization — the process of removing sectioning, and therefore should be placed in a paraffin wax from the tissue once properly fixed on a slide; process before staining 3  ○​ Methods: ​ BASE SLEDGE MICROTOME: Specific type ​ Immersion of slides in xylene of siding microtome in which the movable part ​ Place slides inside the oven — is the block holder oven temp is 55 to 60 degC ​ CRYOSTAT: A refrigerated apparatus used in ​ Pass the slide over a flame fluorescent antibody staining techniques or using alcohol lamp histochemical enzyme studies ​ ULTRATHIN MICROTOME: Tissues SUMMARY QUESTIONS: embedded using plastic resins (for EM) i.e. ​ ROCKING MICROTOME: Type of microtome epoxy must be cut using what type of used to prepare serial sections microtome T ​ FREEZING MICROTOME: Microtome type ​ FREEZING MICRTOME: Type of microtome used to cut tissues to demonstrate neurological invented by Queckette M structures ​ ROTARY MICROTOME: Type of microtome ​ 4-6 micra: thickness of sections produced operated by the rotation of the flywheel, using rotary microtome (range in micra) causing reciprocal motion of knife over the ,R ​ STROPPING: Process of sharpening and block polishing the cutting edge ​ DIAMOND KNIFE: Type of microtome knife ​ BICONCAVE: Type of microtome knife used to used to cut any type of resin block for EM cut paraffin embedded tissues ​ WEDGE ANGLE: It is defined as the angle ​ BEVEL ANGLE: Defined as the angle formed formed by the sides of the knife ES between cutting edges ​ SLIDING MICROTOME: Type of microtome ​ 5-10 deg: Actual angulation in degrees of developed by Adam clearance angle to prevent uneven sections ​ 10-12 micra: Thickness of sections produced ​ GLYCERINE: Mayer’s egg albumin is prepared using rocking microtome by adding equal amounts of egg white and ​ 10-15 micra: Celloidin sections are usually cut EL _______ between _________ thickness ​ CHUCK: Block holder is also called ______ ​ FINE CARBORUNDUM: Type of hone used for ​ CRYOSTAT and FREEZING MICROTOME: badly nicked knives Types of microtome which can be used to ​ 10-20 STROKES: In honing, when using plane G prepare frozen sections wedge knife, the knife is turned over as to ​ GLASS KNIFE: Type of microtome knife used sharpen the other surface every _____ strokes for trimming and semi-thin sectioning of tx ​ FREEZING MICROTOME: Type of microtome AN blocks for EM used to cut tx with heat sensitive structures ​ ROCKING MICROTOME: Type of microtome ​ 0.5 micra: Thickness of sections produced invented by Trefall using ultrathin microtome ​ BELIGIUM YELLOW: Type of hone that gives ​ ROCKING MICROTOME: Type of microtome the best result that has restriction as to the size of block that ​ 40 to 120 double strokes: Number of strokes can be cut required when doing stropping ​ TO PREVENT GROWTH OF MOLDS: ​ ROCKING MICROTOME: The type of Purpose of adding thymol crystals in mayer’s microtome that when used will make block egg albumin re-orientation difficult 4  ​ PLANE WEDGE: Type of microtome knife used to cut tough specimens embedded in paraffin blocks ​ ULTRATHIN MICROTOME: Type of microtome required for tissues fixed using osmium tetroxide (for EM) ​ 27-32 deg: Bevel angle is set at what angulation ​ OILING USING VEGETABLE OIL: Must be done to leather strop prior to use T ​ 45-50 degC: Actual temperature of float out bath M ​ STANDARD SLIDING MICROTOME: Specific type of Sliding microtome regarded as the most dangerous ,R ​ LUBRICANTS FOR HONING: Mineral oil clove oil, xylene, liquid paraffin & soapy water ​ STROPPING: To remove burrs and other irregularities ​ METHODS FOR PREPARING FROZEN ES SECTIONS: Cold knife procedure & Cold Microtome procedure/Cryostat procedure ​ FREEZING AGENTS FOR FREEZING MICROTOME: Liquid nitrogen, isopentane cooled by liquid nitrogen, CO2 gas & aerosol EL sprays ​ FREEZE DRYING: Special way of preserving tissues by rapid freezing (quenching) of fresh tissues at -160 degC G CHAPTER READINGS: INFILTRATION AND SECTIONING AN 5