6. Wash the tissue at least three times for 5 min with methanol by
removing and applying the solution to the tube using a pipette,
without touching the tissue.
7. Transfer the tissue using a tweezer to a sieve, which is standing
in a glass petri dish half filled with ethanol (see Note 13).
8. Wash the tissue containing sieves two times for 5 min with
ethanol by exchanging the liquid in the glass petri dish (see
Note 14).
For the following steps, exchange the liquid in the glass petri
dish containing the sieve. Take care that the entire liquid is removed
from the petri dish before applying the next solution, but do not
apply the solution into the sieve, as this may lead to damage or loss
of the tissue sample.
3.5 Degradation
of Endogenous
Peroxidase
This step is only required if the HRP/TSA detection system shall be
applied:
1. Incubate the sieves for 1 h in freshly prepared 3% (v/v) H 2 O 2 /
ethanol.
2. Wash the sieves twice for 5 min with ethanol.
3.6 Permeabilization
of the Tissue
1. Incubate the sieves in HistoClear/ethanol (1/1) solution for
30 min.
2. Wash the sieves three times each for 5 min with ethanol (see
Notes 15 and 16).
3. Incubate the sieves in 75% and 50% (v/v) ethanol followed by
incubation in 25% (v/v) ethanol/PBS for 15 min each.
4. Wash the sieves with PBS-T for 5 min.
5. Cross-link the tissue for 20 min by changing the liquid in the
glass petri dish to fixative.
6. Wash the sieves twice with PBS-T for 10 min each.
7. Incubate the sieves in freshly prepared pre-warmed
150–400 μg/mL proteinase K solution for 15 min at 37
C
to degrade proteins bound to RNA (see Note 17).
8. Stop reaction by exchanging the liquid against glycine solution
and incubate for 5 min.
9. Wash the sieves twice in PBS-T for 10 min each.
10. Cross-link the tissue for 20 min by sieve incubation in fixative.
11. Wash the sieves twice in PBS-T for 10 min each.
3.7 Hybridization
of Probes
1. Incubate the sieves in pre-hybridization mix for 10 min and
60 min at the desired hybridization temperature (see Note 18).
2. Prepare the hybridization mix: Add freshly denatured fragmentized salmon sperm DNA (denatured for 10 min at 95
C) to a
12
Thomas Dresselhaus and Andrea Bleckmann
removing and applying the solution to the tube using a pipette,
without touching the tissue.
7. Transfer the tissue using a tweezer to a sieve, which is standing
in a glass petri dish half filled with ethanol (see Note 13).
8. Wash the tissue containing sieves two times for 5 min with
ethanol by exchanging the liquid in the glass petri dish (see
Note 14).
For the following steps, exchange the liquid in the glass petri
dish containing the sieve. Take care that the entire liquid is removed
from the petri dish before applying the next solution, but do not
apply the solution into the sieve, as this may lead to damage or loss
of the tissue sample.
3.5 Degradation
of Endogenous
Peroxidase
This step is only required if the HRP/TSA detection system shall be
applied:
1. Incubate the sieves for 1 h in freshly prepared 3% (v/v) H 2 O 2 /
ethanol.
2. Wash the sieves twice for 5 min with ethanol.
3.6 Permeabilization
of the Tissue
1. Incubate the sieves in HistoClear/ethanol (1/1) solution for
30 min.
2. Wash the sieves three times each for 5 min with ethanol (see
Notes 15 and 16).
3. Incubate the sieves in 75% and 50% (v/v) ethanol followed by
incubation in 25% (v/v) ethanol/PBS for 15 min each.
4. Wash the sieves with PBS-T for 5 min.
5. Cross-link the tissue for 20 min by changing the liquid in the
glass petri dish to fixative.
6. Wash the sieves twice with PBS-T for 10 min each.
7. Incubate the sieves in freshly prepared pre-warmed
150–400 μg/mL proteinase K solution for 15 min at 37
C
to degrade proteins bound to RNA (see Note 17).
8. Stop reaction by exchanging the liquid against glycine solution
and incubate for 5 min.
9. Wash the sieves twice in PBS-T for 10 min each.
10. Cross-link the tissue for 20 min by sieve incubation in fixative.
11. Wash the sieves twice in PBS-T for 10 min each.
3.7 Hybridization
of Probes
1. Incubate the sieves in pre-hybridization mix for 10 min and
60 min at the desired hybridization temperature (see Note 18).
2. Prepare the hybridization mix: Add freshly denatured fragmentized salmon sperm DNA (denatured for 10 min at 95
C) to a
12
Thomas Dresselhaus and Andrea Bleckmann