Biology Reference
In-Depth Information
2.2 Plaque Assay
1. Neutral Red staining (classic method): 0.03 % Neutral red
solution in PBS. Vortex vigorously to dissolve well as neutral
red forms crystals if not dissolved properly.
2. Thiazolyl Blue Tetrazolium Bromid (MTT) staining: 5 mg/
mL stock solution of MTT in PBS or DMEM 5 % FBS.
1. Takara PreMix 2× (cat #: RR039A).
2. Hexon primers and probe: Ad18852 (sequence 5
2.3 qPCR
′
-CTTCGATGA
TGCCGCAGT G-3
′
) stock 10
μ
M; Ad18918R (sequence
5
′
-GGGCTCAGGTAC TCCGAGG-3
′
) stock 10
μ
M, probe
5
-FAM-TTACATGCACATCTCGGGCCAGGAC-
TAMRA-3
′
′
. stock 10
μ
M.
2.4
In Vivo Studies
1. Tissue adhesive: Vetbond 3 M, 3 M Animal Care Products,
St. Paul. MN.
2. Primary anti-adenovirus antibody: Rabbit polyclonal Abcam
ref. Ab6982.
3. Secondary antibody: Envision System. Labeled Polymer-HRP
anti-rabbit, DAKO Cytomation.
4. 10× citrate sodium solution: For 500 mL mix 1.9 g citric acid
in 90 mL sterile water and 12.05 g sodium citrate in 410 mL
sterile water.
5. DAB (3,3
′
Diaminobenzidine) buffer: 1,000
μ
L PBS + 4
μ
L
hydrogen peroxide.
6. DAB staining mix: 20
L of DAB chromogen (DAKO) solu-
tion mixed with 1 mL of DAB buffer.
7. ACK lysis buffer: 150 mM NH
4
Cl, 10 mM KHCO
3
, 0.1 mM
Na
2
EDTA. Adjust to pH 7.2-7.4 with 1 N HCl. Filter sterilize
and store at 4 °C.
8. FACS Buffer: PBS 5 % FBS, 0.5 % BSA, 0.07 % NaN
3
(Sodium
azide). Filtered with 0.22
μ
m fi lter.
9. Perm Wash (permebilization): PBS with 1 % FCS, 0.1 % NaN
3
,
0.1 % Saponine. Filtered with 0.22
μ
m fi lter.
10. FACS Fixative: 0.89 % NaCl, 1 % PFA. Adjust to pH 7.4 with
NaOH.
11. Intracellular staining fi rst antibody mix:
CD8 PECy5: 1.5
μ
μ
L of 1:10 dilution + 48.5
μ
L of FACS
buffer.
CD4 PerCPCy5.5: 1.5
μ
L of 1:10 dilution + 48.5
μ
L of FACS
buffer.
12. Intracellular staining second antibody mix:
IFNg APC: 1.5
μ
L of 1:10 dilution + 48.5
μ
L of Perm Wash.
