SECTIONING

BEVEL
SECTIONING
 Cutting facet
 Step 9
 Embedded tissue is trimmed and cut into uniformly thin slices
BEVEL ANGLE
using a microtome
 Angle formed between cutting edge
MICROTOM Y  27-23 degrees
 Knife back/slide: maintains bevel angle
 Three essential parts
o Block holder aka Chuck CUTTING ANGLE
o Knife carrier and knife
o Pawl, ratchet wheel, and adjustment screw  15 degrees
 Optimum
FIVE KINDS OF MICROT O ME  Max penetration of tissue and less distortion

 Rocking Microtome (Cambridge Microtome, 1881) CLEARANCE ANGLE

 Rotary Microtome (1885-1886)
 0-15 degrees
 Sliding Microtome (1789-1798)
 Angel formed between the surface of the block and cutting edge of
 Freezing Microtome (1848)
the knife
 Ultra-Thin Microtome
 Other Microtome RAKE ANGLE

MICROTOME KNIVES  Angle of the upper facet and surface of tissue block

 Mostly made of stainless steel WEDGE ANGLE

 Angle of the sides of knife

MICROTOME KNIVES
Plane Concave  Length: 25 mm HONING AND STROPPING
 Shortest
 Less concave for Celloidin HONING
 More concave side for  Knife hard sharpening
paraffin
 Process of removing Nicks (Irregularities of the knife)
 Use: Base Sledge, Rotary or
 Movement: Heel to toe
Rocking Microtome
 Number of stroke per surface: 10-20 strokes
Biconcave Knife  Length: 120 mm
 Longest HONES /OIL STONE
 Both sides are biconcave  Dimension: 8 inches x 3 inches
 Cutting paraffin sections
 Use: Rotary Microtome  Types:
o Belgium Yellow
Plane Wedge  Length: 100 mm  Best result
 For frozen sections or very
o Arkansas
hard tissues
 Use: Base Sledge or Sliding o Fine Carborundum
Microtome  Badly nicked knives
o Plate Glass
OTHER TYPES OF KNIVE S  Excellent substitute or alternative for oil
stone (last resort)
DISPOSABLE BLADE
LUBRICANTS
 More commonly used
 Coated with polytetrafluoroethylene  To moisten stone
 Types:
GLASS AND DIAMOND KNIVES o Soapy Water
 Most commonly used
 For Ultra-Thin Microtome o Mineral Oil
 Glass Knives or Ralph Knives o Clove Oil
o Xylene
o Liquid Paraffin

VILLAPANDO, MARK ANDREW OBEJERA 1

 SECTIONING

KNIFE SHRPENERS CLEARING AGENTS USED

 Mechanical honing  Glycerin and gum syrup are used (water soluble)

Note: clean the honed knife with xylene NO NEED FOR DEALCOHOLIZATION

 Function of clearing agent: increased refractive index

STROPPING
MOUNTING
 Polishing
 Removal of burrs (irregularities of knife formed after honing)  Use of synthetic water-soluble glycols and resins
 Movement: Toe to heel  Optimal cutting temperature compound
 Uses Paddle Stroke  Tissue Block: 2-4 mm
o Horse leather  Tissue section can be cut from 5-10 um
 Dimension: 3-4 inches to 18 inches
 Should be oiled on the back DIFFICULTIES DUE TO FAULTY FIXATION,
o Cannot use mineral oil DEHYDRATION, CLEARING, AND EMBEDDING
 Double stroking
o 40-120 strokes  Brittle; hard tissue
o Toe-toe, heel-heel o Over dehydrated
 Clearing agent become milky
o Not totally dehydrated
TYPES OF SECTIONS
 Soft and mushy tissue
o Under fixed
PARAFFIN SECTION
 Tissue becomes opaque
 4-6 um o Not enough clearing
 Average: 5 um  Very hard tissues; tissue shrinkage
 Air holes on trimmed tissue block
CELLOIDIN SECTION  Moist block that tends to crumble
 Tissue block smells like xylene
 10-15 um o Not proper infiltration

RENAL BIOPSY SECTION

FISHING O UT
 2 um
 Place the ribbon of section into the water bath for it to flatten
SEMI-THIN SECTION o Product of sectioning
o 30 seconds time it takes for the ribbon to flatten
 Using plastic embedding medium o Do not let the ribbon stay on the water bath for more
than 1-2 minutes
ULTRA-THIN SECTION (EM)  Separate the ribbon of sections into individual sections using
forceps
 Recommended section: 80 nm (silver or straw-colored sections)
 Start fishing out each section using slide (lightly smeared with
adhesive)
FROZEN SECTION (CRYOSTAT)
 Immerse the slide vertically
 4 or 10-15 um section if microtome used is rotary  Fish out a section
 Gently lift the side vertically
FROZEN SECTION  Slide is then stood to drain at an angle of 60-85 O for 2-5 minutes
 Dry the sections using either of the following methods:
 For rapid diagnosis o Hot plate 45-55 OC for 30-45 minutes
 For enzyme histochemistry o Leaving it in an incubator overnight
 Demonstration of soluble substances  Best for nervous tissue
 Immunofluorescence and immunocytochemical stain o Wax oven 56-60 OC for 2 hours
 Specialized silver stain o In a blower type electric slide dryer for 20-30 minutes
with 50-55 OC
o Above a bunsen burner until the wax melts
FROZEN SECTION
 Urgent method
Cold Knife  Coolness use of conventional freezing microtome
 Tissue Block: 3-5 mm
 Dew line
o Point in which sections may be cut at 10 um;
frozen tissue starts to thaw (with the finger) and
become visible to the naked eye
 Use of camel hairbrush
o Used to remove the ribbon that sticks to the knife
blade
Cryostat  Use of cryostat
 Optimum temperature: -18 to -20 OC
 Average: -20 OC

VILLAPANDO, MARK ANDREW OBEJERA 2

 SECTIONING

DIFFICULTIES DURING SE CTIONING FOUR STAGES TO FREE Z E DRYI NG

 Mushy tissue blocks that lead to sections to crumble and feather

QUENCHING
o Remedy: Decalcification then ice
 Tearing of tissue from block  Stops the chemical reaction and diffusion by rapid freezing
 Section fails to form ribbon
 Section rolls up on cutting that they adhere; wrinkled or jammed DRYING
 Sections adhere knife or other parts of microtome
 Most time consuming
 Chatter – horizontal or parallel lines across the section
 Floater and contaminants FIXATION

TISSUE ADHESIVES

VAPOR FIXATION
ALBUMIN
 Most important: Formalin
 Mayer’s egg albumin
 Most common
 Egg whites, glycerol, thymol crystal FREEZE SUBSTITUTION
 Disadvantage: background is also stained; may take up the  Similar to freeze drying except without vacuum procedure
albumin
 Makes use of Rossman’s formula
o 1% acetone and dehydrated in absolute alcohol
GELATIN
 Routine paraffin wax impregnation and embedding is done
 Provides firmer attachment than albumin
 Disadvantage: Stain with many dyes

CELLULOSE

 Used in the form of 1% methyl cellulose

 Disadvantage: Not staining to any appreciative histochemical
reagent

POLY-L-XYLENE

 Use as general purpose for section adhesion

 Used for adhesion of immunohistochemistry
 Disadvantage: Not production of background while staining

SODIUM SILICATE

 Commercial syrup with 1:10 dilution

 Advantage: has strong adhesive properties, has tendency to stain
most of the dyes but cannot be affected by mild alkaline solution

RESIN

 Has greatest adhesion

 Made up of epoxy
o Araldite – made of epoxy resins
 Dilute to 1:10 acetone

SPECIAL PRO CESSI NG T ECHNIQ UES

 For histochemical evaluation (Enzyme studies)

FREEZE DRYING

 Preserve at 60 OC
o Rapid freezing (quenching)
 Desiccation by transferring the frozen tissue in a vacuum at -40 OC
 2 mm tissue thickness

VILLAPANDO, MARK ANDREW OBEJERA 3