Frozen Section Techniques Flashcards
method of processing when rapid diagnosis must be made or when a staining technique does not work on routinely processed tissue
frozen section
demonstrates fat, enzyme IHC (muscle), immunofluorescence
frozen section
4 things to obtain a quality section
-good freezing technique
-cutting temperature for specific specimens
-use of anti roll plates
-proper embedding
cryobar/platform temperature should be around
-30 to -50 deg celcius
speeds up freezing and provides a flat surface to cut
heat extractor
keep this cryoembedding media cold
OCT
check this on the bottle of OCT
temperature
keep these in the cryostat
chucks
steps of making frozen block (5)
-put OCT on chuck
-orient tissue
-put more OCT
-apply heat extractor
-cut
non fatty tissues cut between
-15 to -20 deg celcius
breast, skin, adrenal, nervous tissue cut at this temp
-30 deg celcius
you should have a border of this around the tissue (except in muscle)
OCT
why might the section shred as it comes off the knife (3)
-knife edge damaged
-tissue material not suitable for temperature
-tissue has suture/staples/calcium deposits
how can you fix the section shredding as it comes off the knife (2)
-clean knife edge or change it
-decrease/increase temperature
why might the section bunch up at the knife edge or not slide off smoothly (4)
-blade holder is dirty or iced up
-blade is too warm
-knife angle
-dull blade
how can you fix the section bunch up at the knife edge or not slide off smoothly (4)
-clean blade holder with 100% ethanol to remove ice
-cool the blade
-adjust the angle
-move the knife
why might the section have thick and thin sections (5)
-blade is loose
-holder is loose
-block is loose
-blade is dull
-microtome not advancing properly
how can you fix the section have thick and thin sections (2)
-check all of the reasons why it might
-defrost disinfect and lubricate instrument
provides a rapid nuclear stain for frozens
thionin
good for demonstrating Nissl substance
thionin
demonstrates neutral lipids in frozen sections
oil red o
differentiates between type I, type IIA, type IIb fibers, and helps separate myopathic from neuropathic processes
ATPase stain
extremely susceptible to ice crystal artifact which can make stain uninterpretable
muscle tissue
use this to orient muscle and embed on a cork
tragacanth gum
after you cut tissue, you must fix the slide in formalin or isopropyl ETOH for this stain
oil red o
steps of oil red o (6)
-stain in Oil red O for 10 mins
-wash in tap water
-stain in hematoxylin for 20s-1min
-wash
-blue in ammonia water
-mount with aqueous mountain media
steps of thionin staining (4)
-briefly fix slide in 95% ETOH
-drop thionin stain over slide for 10 sec
-drain on paper towel and gently rinse in tap water
-wet mount slide
thionin stained slides may be permanently coverslipped by (3)
removing wet coverslip, immerse slides in 10% formalin, and stained with basic H&E
ideal temp of isopentane for freezing muscle biopsies to prevent ice crystals
-150 deg celcius
