Biology Reference
In-Depth Information
25 ml Secondary antibody diluent example:
●
1% GS = 250 ml
2% BSA = 0.5 g
0.3% TX = 75 ml
0.5 M PBS = 24.6 ml
Goat anti-rabbit FITC = 62.5 ml
10. Rinse: 0.1 M PBS 3× 10 min
11. 0.1 M PB 6× 10 min
12. Mount sections: Place sections on gelatin-coated slides directly
from 0.1 M PB. Tilt slide to remove excess buffer, and dab
with Kimwipe to further remove excess PB. Let air dry ~1 h.
Then, cover slip the slide with a few drops of mounting media
(i.e., aquamount or DPX). Dry slides overnight at RT. Finish
by sealing cover slip edge with clear nail polish.
1. Preparation of microcarriers
Evenly distribute gold microcarriers (50
3.4. Diolistic Labeling
Using a Gene Gun
(Adapted from Ref. (
34
) )
mg; 1 mm diame-
ter) on a glass slide and 0.125 mg of DiI or DiO dissolved
in methylene chloride. See Note 8.
Allow the methylene chloride to fully evaporate (~5 min).
●
●
Gently scrape the dye-coated gold particles on a piece of
●
fi lter paper and then place in test tube. Resuspend the par-
ticle in 3 ml of sterile dH
2
O.
Sonicate the dye slurry for 5 min to prevent the formation
●
of clusters.
2. Loading the microcarrier suspension into bullets using the
tubing prep station
Dry a 75-cm piece of Tefzel tubing by purging with a
●
steady fl ow of nitrogen (0.3-0.4 LPM) for 15 min.
Insert the PVP solution into the tubing using the tubing
●
preparation station and leave for 2 min. Remove the PVP
solution using a syringe attached to the end of the
tubing.
Allow the tubing to air-dry (~10 min).
●
Prior to loading, vortex the dye slurry to ensure an even
●
suspension.
Use the tubing preparation station to insert the gold
●
suspension into the Tefzel tubing by attaching a 10-ml
syringe to the end of the tubing, inserting the other end
into the tubing prep station. Make sure tubing is in hori-
zontal position and passes through the o-rings of the
tubing prep station.
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