Biology Reference
In-Depth Information
2. Cool down paraffi n blocks at −20 °C and cut sections of 4
m
of thickness. Once cut, sections are extended in distilled water
at 42 °C. For better adhesion use poly-l-lysine slides and deion-
ized water in the water. Fish the slices on the slides and dry at
37 °C for 30 min. Put two paraffi n sections per slide in order
to have a control without the specifi c antibody.
3. Deparaffi nize and hydrate tissue sections as follows: heat slides
for 2 h at 65 °C or overnight at 37 °C. Wash four times for
5 min in xylene; three times for 5 min in 100 % ethanol; three
times for 5 min in 96 % ethanol; and once for 5 min in 70 %
ethanol. Rinse sections once for 5 min in deionized water.
4. Block endogenous peroxidase incubating sections for 10 min
in 0.33 % hydrogen peroxide. Rinse once for 5 min in deion-
ized water and three times for 5 min in PBS pH 7.4.
5. Antigen retrieval: in a pressure cooker submerge slides in 1×
citrate sodium solution prepared in PBS and adjusted to
pH = 6. Heat to reach maximum pressure, wait 2 min, and stop
heating. Wait about 20 min until pressure cooker cools down.
Rinse slides once for 5 min in deionized water and three times
for 5 min in PBS pH 7.4.
6. Block sections with 20 % goat or horse serum in PBS for
30 min at RT in a sealed humidity chamber.
7. Remove the serum and cover sections with primary anti-
adenovirus antibody (e.g., rabbit polyclonal) 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.
8. Wash the sections three times for 5 min in PBS + 0.05 % Tween
20.
9. Without letting sections dry out, add the secondary antibody
(HRP-conjugated anti-mouse or anti-rabbit) and incubate for
30 min at RT in a humidity chamber.
10. Rinse sections three times for 5 min in PBS + 0.2 % Triton
X-100.
11. Cover sections with DAB staining mix and incubate until a
brown precipitate appears (2-5 min). Stop reaction by rinsing
sections with tap water.
12. Counterstain with hematoxylin for 1 or 2 min. Rinse slides
about 10 min with tap water.
13. Dehydrate once for 2 min in 70 % ethanol, three times for
2 min in 96 % ethanol, three times for 2 min in 100 % ethanol,
and four times for 5 min in xylene.
14. Mount slides with DPX.
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