1C. Procedures:
Deeply anesthetize mice (e.g. sodium pentobarbital 80–100 mg/kg IP, no
response to firm forepaw pinch), surgically open the chest to expose pericardial
region, cut the right atrium, and insert a perfusion cannula into the left ventricle.
We use gravity (i.e. pressure-based) flow, aiming for *3 ft hydrostatic pressure.
A peristaltic pump also works; most important is technical success, getting fix to
the brain reasonably quickly so the body and brain harden properly. After a brief
(*30 s) flush with normal saline, run fixative for *10 min. Remove the brain,
put it in the same fixative, store overnight at 4 °C, then replace with buffer and
keep in the refrigerator. Fixed brains are stable in the refrigerator for >1 week.
Collect fixative for hazardous waste disposal. Procedure should take place in a
hood or with personnel-protecting ventilation, as fumes are hazardous.
2. Tissue blocking, mounting, sectioning
2A. Required materials and equipment:
Double-edged razor blades
Tweezers/forceps
Cyanoacrylate cement (we use “Crazy Glue”)
70–95% ethanol in dispensing bottle (for cleaning blades and Vibratome)
Vibratome and mounting blocks
Saline and saline ice cubes
Paint brushes, or other section pickers
Collection trays (24-well cell culture trays work well)
Tray sealers (adhesive plastic film) minimize buffer loss and tissue dehydration
0.025% sodium azide in 0.01 M PBS (only for long storage, do not allow contact
with metal, collect for disposal as hazardous waste)
Petri dish and/or dental wax
2B. Preparations:
0.01 M PBS (store in refrigerator)
0.2 g sodium phosphate monobasic, monohydrate
2.6 g sodium phosphate dibasic, heptahydrate
8.8 g sodium chloride
1 L distilled or deionized water
Sodium azide storage solution (collect used solutions as hazardous waste)
0.25 g sodium azide (use non-metal spatula)
1 L 0.01 M PBS
2C. Procedures:
Remove brain from storage vial and place in a petri dish containing buffer or
PBS. Use tweezers or fine forceps under a dissecting microscope to peel and
remove as much remaining vasculature and pial membrane as you can. Using a
6 Conjugate Immunofluorescence—SEM Array Tomography …
167
Deeply anesthetize mice (e.g. sodium pentobarbital 80–100 mg/kg IP, no
response to firm forepaw pinch), surgically open the chest to expose pericardial
region, cut the right atrium, and insert a perfusion cannula into the left ventricle.
We use gravity (i.e. pressure-based) flow, aiming for *3 ft hydrostatic pressure.
A peristaltic pump also works; most important is technical success, getting fix to
the brain reasonably quickly so the body and brain harden properly. After a brief
(*30 s) flush with normal saline, run fixative for *10 min. Remove the brain,
put it in the same fixative, store overnight at 4 °C, then replace with buffer and
keep in the refrigerator. Fixed brains are stable in the refrigerator for >1 week.
Collect fixative for hazardous waste disposal. Procedure should take place in a
hood or with personnel-protecting ventilation, as fumes are hazardous.
2. Tissue blocking, mounting, sectioning
2A. Required materials and equipment:
Double-edged razor blades
Tweezers/forceps
Cyanoacrylate cement (we use “Crazy Glue”)
70–95% ethanol in dispensing bottle (for cleaning blades and Vibratome)
Vibratome and mounting blocks
Saline and saline ice cubes
Paint brushes, or other section pickers
Collection trays (24-well cell culture trays work well)
Tray sealers (adhesive plastic film) minimize buffer loss and tissue dehydration
0.025% sodium azide in 0.01 M PBS (only for long storage, do not allow contact
with metal, collect for disposal as hazardous waste)
Petri dish and/or dental wax
2B. Preparations:
0.01 M PBS (store in refrigerator)
0.2 g sodium phosphate monobasic, monohydrate
2.6 g sodium phosphate dibasic, heptahydrate
8.8 g sodium chloride
1 L distilled or deionized water
Sodium azide storage solution (collect used solutions as hazardous waste)
0.25 g sodium azide (use non-metal spatula)
1 L 0.01 M PBS
2C. Procedures:
Remove brain from storage vial and place in a petri dish containing buffer or
PBS. Use tweezers or fine forceps under a dissecting microscope to peel and
remove as much remaining vasculature and pial membrane as you can. Using a
6 Conjugate Immunofluorescence—SEM Array Tomography …
167