Biology Reference
In-Depth Information
6. Analyze the amplifi cation curves (threshold cycle to obtain
positive signal or crossing point) to obtain de virus DNA con-
centration of the sample.
3.6 Immunohisto-
chemistry with Frozen
Tissue Sections
1. Embed fresh tissues carefully in OCT in plastic cryomold, tak-
ing care not to trap air bubbles surrounding the tissue. Freeze
tissue by setting the mold on dry ice. The frozen blocks may be
stored at −80 °C for a long term.
2. Before cutting sections, allow frozen blocks to equilibrate at
−25 °C in the cryostat chamber for about 5 min.
3. Cut 5
m sections on slides, placing two or three separated
sections per slide. For better adhesion use poly-l-lysine slides.
Slides can be stored at 4 °C for a few days or at −80 °C long
term.
4. To stain, let the sections dry for at least 30 min at RT and fi x
them in 4 % formaldehyde for 10 min at RT.
5. Rinse sections three times for 2 min in deionized water and
once for 10 min with PBS + 0,05 % Tween 20.
6. Carefully dry slides with paper and encircle sections using a
water repellent marker (PAP PEN).
7. Block sections with 20 % goat or horse serum in PBS for
30 min at RT in a sealed humidity chamber.
8. Remove the serum and cover sections with primary anti-
adenovirus antibody (e.g., rabbit polyclonal Ab6982) diluted
1/100 in PBS + 0.05 % Tween 20. Incubate 1 h at RT in a
humidity chamber. For a negative control incubate one section
of each slide with PBS or with a unspecifi c rabbit antibody
diluted 1/100 in PBS + 0.05 % Tween 20.
9. Wash the sections three times for 5 min in PBS + 0.05 % Tween 20.
10. Without letting sections dry out, add the secondary antibody
(e.g., anti-rabbit labeled with fl uorescence) diluted according
the provider in PBS + 0.05 % Tween 20. Incubate as before for
at least 1 h.
11. Wash the sections three times for 5 min in PBS + 0.05 % Tween
20.
12. Mount coverslips with mounting media, DAPI, and antifading
agents, and observed the stained sections in a fl uorescence
microscope.
μ
1. Slice (3 mm width) tumor and organs and place them inside
tissue cassettes. Fix tissue in buffered formalin or fresh 4 %
paraformaldehyde during 48 h at RT. Dehydrate and embed in
paraffi n (70 % ethanol twice 1 h; 80 % ethanol twice 1 h; 95 %
ethanol twice 1 h, 100 % ethanol, three incubations of 1 h;
xylene three incubations of 1 h; and paraffi n wax at 58 °C two
changes of 1 h.).
3.7 Immunohisto-
chemistry on Paraffi n-
Embedded Tissue
Sections
