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MICROTECHNIQUE VI.

Embedding
PRELIMS REVIEWER A. Orient tissue
1. Cross section
II. Fixation: preservation of tissue structure 2. Longitudinal section
A. Avoid autolysis B. Dissection orientation
B. Common fixatives: C. Avoid bubbles
1. Formaldehyde, buffered D. Procedure
2. glutaraldehyde 1. Place tissue cassette in melted paraffin
3. 70% alcohol 2. Fill mold with paraffin
4. Heat: boiling water, microwave 3. Place tissue in mold
C. Application of fixative 4. Allow to cool
1. Immersion
2. Perfusion
a. intracardiac perfusion
3. Sample size considerations
4. Exposure time
D. Procedure
1. Dissection
2. Trimming and orientation
3. Immersion fixation
a. tissue cassette

VII. Sectioning – Trimming the Block


Untrimmed tissue block
Trimmed block with excess paraffin removed and block face in a
trapezoid shape

III. Dehydration
A. Definition: removal of water
B. Rationale: for paraffin embedding/sectioning
C. Steps
1. Wash out fixative A. Rotary microtome
2. graded series of alcohol 1. 5-10 µm
a. 70%, 95%, 100%, 100% 2. Resolution vs. staining
3. Replace water by diffusion B. Cryostat
4. Not too long, not too short C. Freezing microtome
D. Procedure D. Vibratome
1. Automatic tissue processor E. Procedure
a. overnight 1. Place tissue block in microtome with wide edge of trapezoid
2. Baths: water, 70, 95, 100,100 % alcohol lowest, and parallel to knife
3. Clearing agent: 2 baths of xylene 2. Advance blade toward block
3. Begin sectioning
IV. Clearing
A. Paraffin solvent
B. Xylene, “clearing agent”
C. Makes tissue appear “clear”

V. Infiltration
A. Replace xylene with paraffin
B. Immerse in melted paraffin
1. ~55oC MP
C. Remove all bubbles, xylene
D. Procedure NOTE: Many of the figures in the text are of plastic embedded
1. Two baths of melted paraffin sections cut at 1 µm thickness, and thus showing better
resolution than 5-10 µm paraffin sections seen in lab.
VIII. Mounting sections XI. Pitfalls
A. Poor fixation (poor structural details)
B. Inadequate dehydration
C. Contaminated xylene (milky)
D. Poor infiltration (bubbles, poor support)
E. Embedding: orientation, bubbles
F. Poor sectioning
1. Knife marks (scratches perpendicular to knife edge)
2. Compression (waves parallel to knife edge)
G. Mounting sections
A. 40oC water bath 1. Folds & tears
1. Flattens paraffin section 2. Excess albumin (stain)
2. Permits mounting on slide H. Staining
B. Gelatin & albumin 1. Inadequate rehydration (uneven staining)
C. Glass slides 2. Too dark or too light (timing off)
D. Oven / air dry 3. Inadequate agitation
I. Coverslipping
IX. Staining 1. Bubbles
A. Basic dye: hematoxylin J. Coverslipping
1. Basophilic structures: DNA, RNA 2. Excess Permount
2. Differentiation: sodium bicarbonate 3. Two coverslips
B. Acid dye: eosin
1. Acidophilic (eosinophilic) structures XII. Interpretation
a. mitochondria, collagen A. Artifacts
C. Water soluble dyes (paraffin sections) B. 3D from 2D
D. Clearing agent (remove paraffin)
E. Rehydrate
F. Stain (trial & error timing)

NOTE: most figures in the text are not stained with H & E,
unlike the slides in our collection (and most collections).

G. Procedure

1. Slide rack
2. Solutions
a. rehydration
b. stain
c. dehydration

X. Cover slipping
A. Coverslip & mounting medium (not miscible with water)
B. Dehydrate
C. Clearing agent
D. Permount