MICROTECHNIQUEPRELIMS REVIEWER II. Fixation: preservation of tissue structure
A. Avoid autolysis B. Common fixatives:
1. Formaldehyde, buffered2. glutaraldehyde3. 70% alcohol4. Heat: boiling water, microwave
C. Application of fixative
1. Immersion2. Perfusiona. intracardiac perfusion3. Sample size considerations4. Exposure time
D. Procedure
1. Dissection2. Trimming and orientation3. Immersion fixationa. tissue cassette
III. Dehydration
A. Definition: removal of water B. Rationale: for paraffin embedding/sectioning C. Steps
1. Wash out fixative2. graded series of alcohola. 70%, 95%, 100%, 100%3. Replace water by diffusion4. Not too long, not too short
D. Procedure
1. Automatic tissue processor a. overnight2. Baths: water, 70, 95, 100,100 % alcohol3. Clearing agent: 2 baths of xylene
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. ~55
o
C MP
C. Remove all bubbles, xylene D. Procedure
1. Two baths of melted paraffin
VI. Embedding
A. Orient tissue
1. Cross section2. Longitudinal section
B. Dissection orientationC. Avoid bubbles D. Procedure
1. Place tissue cassette in melted paraffin2. Fill mold with paraffin3. Place tissue in mold4. Allow to cool
VII. Sectioning – Trimming the Block
Untrimmed tissue block Trimmed block with excess paraffin removed and block face in atrapezoid shape
A. Rotary microtome
1. 5-10
µ
m2. Resolution vs. staining
B. Cryostat C. Freezing microtome D. Vibratome E. Procedure
1. Place tissue block in microtome with wide edge of trapezoidlowest, and parallel to knife2. Advance blade toward block 3. Begin sectioning
NOTE: Many of the figures in the text are of plastic embedded sections cut at 1 µm thickness, and thus showing better resolution than 5-10 µm paraffin sections seen in lab.
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