Biology Reference
In-Depth Information
Following the TN rise, block slides prior to antibody incubation at 37
Cin30mL
blocking buffer (BB) for 30 min.
Add 10
m
L of anti-Digoxigenin-AP Fab fragments (Roche, #11093274910) to
30 mL fresh BB and incubate for 3 h at 37
C.
C. Rinsing and Signal Development
Rinse slides in the following buffers:
1) 10 min - TN (room temperature)
2) 10 min - TN (room temperature)
3) 10 min - TNM pH 9.5 (room temperature)
4) Developer - incubate with slides overnight in the dark (e.g., by covering entire
Coplin jar in aluminum foil) at room temperature.
The next day, rinse twice in TN-EDTA for 10 min each, and add mounting medium
(e.g., Vectashield, Vector Labs). Cover with 22
40 mm coverslip, wipe off excess
mounting medium, and seal the coverslip with clear nail polish. Slides can be kept up
to 6 months at 4
C, but for best image quality observe within a week. After several
weeks, a colored precipitate will begin to form.
VI. Materials
1. DEPC-ddH
2
O (0.1% v/v)
Millipore water or equivalent
500 mL
DEPC (Sigma)
0.5 mL
Suspend the DEPC by shaking vigorously, leave in a fume hood over night, and
autoclave (121
C at 15 lb/in
2
for 15 min).
2. NTF
2-bromo-2-nitro-1,3-propanediol (Fisher, # AC15882-1000)
30 g
Diazolidinyl Urea (MP Biomedical, # ICN19019183)
30 g
Zinc Sulfate Heptahydrate, ZnSO
4
7H
2
O
12 g
Sodium Citrate (anhydrous), Na
3
C
6
H
5
O
7
2.9 g
Add DEPC-ddH
2
O up to 1 L. Make and store in an autoclaved glass container at
room temperature. Heat 30-50 mL to 37
C in a Coplin jar prior to use (Of this
heated aliquot, do not add unused fixative back to stock, and do not reuse.).
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