Biology Reference
In-Depth Information
Remove the supernatant at a constant speed using the
●
syringe to leave a strip of gold.
Rotate the tubing in the tubing prep station for 3-4 min
●
to ensure an even spread of gold in the tubing.
Using the tubing cutter in the tubing prep station, cut the
●
tubing to desired length, typically 0.5″. Dye bullets can be
stored (desiccated and light-protected) at RT for up to
3 months.
3. Particle Delivery (Bombardment) using the Helios Gene Gun
Insert dye bullet cartridges (12 slots available) into the car-
●
tridge holder and place into gene gun. A different cartridge
holder for each dye is recommended.
Attach the gene gun to the helium cylinder in preparation
●
for use. Set the gas pressure (~75 psi for intact tissue using
modifi ed barrel as per (
34
). See Note 9.
Hold the gene gun approximately 3 cm above and perpen-
●
dicular to the target tissue. Activate the safety interlock
switch and press the trigger button to deliver the dye
microcarriers to the tissue. See Note 10.
4. Notes and Tips
4.1. Notes
1. Be sure the sodium phosphates (mono- and dibasic) are anhy-
drous and not hydrated as the formula weights will be different.
2. PFA is a known carcinogen and should be used with extreme
care and proper personal protective equipment. Check with
institutional offi ce(s) of occupational health and safety or
chemical safety for specifi c requirements.
3. Methylene chloride is toxic. Prepare in fume hood and wear
appropriate personal protective equipment. Check with insti-
tutional offi ce(s) of occupational health and safety or chemical
safety for specifi c requirements.
4. Prior training and approval from the Institutional Animal Care
and Use Committee is required.
5. Slower fl ow rates are preferred as perfusion should be thought
of as a dialysis process exchanging water and formaldehyde
rather than a fl ushing procedure.
6. Brain tissue will sink into the sucrose solution indicating that
sucrose has fully infi ltrated the brain and cryoprotection is
suffi cient.
7. The fl uorescent secondary antibody is light sensitive making
the next steps in the protocol light sensitive components.
Perform in dim light and store tissue samples in dark.
Search WWH ::
Custom Search