21
a)
After the adhesive coating on slide has been polymerized, carefully peel off
the pink Tape.
b)
To avoid uncontrolled melting and/or drying of the section, immediately bring
the room temperature aqueous fixative into the cryostat and immerse the slide
into the fixative. Dip 2-3 times.
c)
Continue to fix for 15 to 30 seconds at room temperature.
d)
Rinse the slide in water and proceed with a chosen staining protocol.
“Anhydrous” Fixation Protocol
-
To be used after “freeze-substitution” in a cold solvent
such as acetone at -30
°
C or colder.
“Anhydrous” fixation preserves
most
cellular components and is highly recommended
for routine stains. Note: Not recommended when ice crystal size is large and widespread
(poor freezing). In such cases the aqueous fixative protocol should be followed. An
“anhydrous” fixative contains a maximum of 30% water.
1.
AN/DMF/Glut
Dimethylformamide (DMF)
35 ml
Acetonitrile (or AN)
35 ml
25% glutaraldehyde
10 ml (50% Glut - 5 ml)
Add H
2
O to make up to
100 ml
Wait at least 1 our before use.
2.
Formal-Ethanol
Ethanol (100% alc.)
86 ml
Formaldehyde
14 ml
Bring the fixative into the cryostat and transfer the freeze-substituted slide directly from
the acetone bath into the fixative. Continue fixation at room temperature for 15 to 30
seconds. (Transfer is made
within
the cryostat to prevent condensation of water on the
cold slide prior to fixation.)
