razor blade, make a block of tissue and glue it to a mounting block using Crazy
Glue (not too much or it will rise into tissue gaps and cause sectioning problems). Avoid getting glue on fingers; if skin glues to block, remove glue with
acetone. Re-immerse in buffer to keep tissue moist.
Set up Vibratome with saline and saline ice cubes or other chilling means. Have
collection trays filled with buffer ± azide on a chilled surface (we often use
shipping cold packs, which we store in a freezer); mounted tissue blocks should
be kept wet and cold until sectioned. For freeze-substitution processing, cut
sections 100–200 µm thick. We typically cut 200 µm for capsule embedment of
small pieces, but if processing an entire section it should be no more than 100 µm
to ensure proper resin cure. It is usually wise to cut some 50 µm sections between
sets of 200s for Nissl-stained reference or other LM/EM purposes. If possible, the
block surface glued to base should be larger than the portion to be cut.
3. Freeze-substitution and embedment in Lowicryl HM-20
3A. Required materials and equipment:
Processing shell vials with caps (1 dram EMS/Fisher) and vial holders (aluminum, EMS #72640), and/or scintillation vials (20 ml)
Fine scalpel blades (#11 or #15)
Small, fine-tipped paint brush
Styrofoam box(es), ± foil lining
Dry ice
100% ethanol (200 proof)
Cold packs
Disposable polyethylene transfer pipets (Fisher #13-711-9CM)
Glycerol
CaCl 2
Sodium acetate trihydrate (ACS)
Uranyl acetate (depleted, available from EMS, Ted Pella, Fisher)
Methanol, (absolute, certified ACS, Fisher Scientific #A412-500)
Lowicryl HM-20 embedding kit (EMS #14340, or Ted Pella)
Freeze-substitution unit (Leica AFS or equivalent) with flow-through capsules
and gelatin capsules for small chunks (1 mm  1–2 mm  200 µm)
For whole sections (100 µm thick), use baskets (Leica #G3314A)
For wafering and curing of sections: glass slides, scoring tool, ACLAR, and
upside-down metal cans for wafer support
ACLAR embedding film (7.8 mil thickness, EMS #50425)
Oven capable of 60 °C for drying things before use
Independent temperature probe for AFS unit (Omega HH800A with Omega
5SC-TT-K-36-36 precision fine wire thermocouple, insulated, self-adhesive)
Acetone (can be used as substitution medium, but must be strictly anhydrous and
doesn’t work as well as methanol in humid climates or conditions); we use
acetone mainly for cleaning.
168
K. D. Micheva and K. D. Phend
Glue (not too much or it will rise into tissue gaps and cause sectioning problems). Avoid getting glue on fingers; if skin glues to block, remove glue with
acetone. Re-immerse in buffer to keep tissue moist.
Set up Vibratome with saline and saline ice cubes or other chilling means. Have
collection trays filled with buffer ± azide on a chilled surface (we often use
shipping cold packs, which we store in a freezer); mounted tissue blocks should
be kept wet and cold until sectioned. For freeze-substitution processing, cut
sections 100–200 µm thick. We typically cut 200 µm for capsule embedment of
small pieces, but if processing an entire section it should be no more than 100 µm
to ensure proper resin cure. It is usually wise to cut some 50 µm sections between
sets of 200s for Nissl-stained reference or other LM/EM purposes. If possible, the
block surface glued to base should be larger than the portion to be cut.
3. Freeze-substitution and embedment in Lowicryl HM-20
3A. Required materials and equipment:
Processing shell vials with caps (1 dram EMS/Fisher) and vial holders (aluminum, EMS #72640), and/or scintillation vials (20 ml)
Fine scalpel blades (#11 or #15)
Small, fine-tipped paint brush
Styrofoam box(es), ± foil lining
Dry ice
100% ethanol (200 proof)
Cold packs
Disposable polyethylene transfer pipets (Fisher #13-711-9CM)
Glycerol
CaCl 2
Sodium acetate trihydrate (ACS)
Uranyl acetate (depleted, available from EMS, Ted Pella, Fisher)
Methanol, (absolute, certified ACS, Fisher Scientific #A412-500)
Lowicryl HM-20 embedding kit (EMS #14340, or Ted Pella)
Freeze-substitution unit (Leica AFS or equivalent) with flow-through capsules
and gelatin capsules for small chunks (1 mm  1–2 mm  200 µm)
For whole sections (100 µm thick), use baskets (Leica #G3314A)
For wafering and curing of sections: glass slides, scoring tool, ACLAR, and
upside-down metal cans for wafer support
ACLAR embedding film (7.8 mil thickness, EMS #50425)
Oven capable of 60 °C for drying things before use
Independent temperature probe for AFS unit (Omega HH800A with Omega
5SC-TT-K-36-36 precision fine wire thermocouple, insulated, self-adhesive)
Acetone (can be used as substitution medium, but must be strictly anhydrous and
doesn’t work as well as methanol in humid climates or conditions); we use
acetone mainly for cleaning.
168
K. D. Micheva and K. D. Phend