John M. S. Bartlett.pdf - Bio-Nica.info

Direct and Indirect In Situ PCR 437
3. Incubate tissue for 30–60 min in 90% ETOH.
4. Incubate tissue for 30–60 min in 96% ETOH.
5. Incubate tissue for 30–60 min in 100% ETOH.
6. Incubate tissue for 30–60 min in 100% ETOH.
7. Incubate tissue for 60 min in xylene100% ETOH (11).
8. Incubate tissue 2 × 60 min in xylene.
9. Incubate tissue 3 × 60 min in paraffin.
10. Allow to cool and harden the paraffin for several hours.
3.4. Preparation of Samples
3.4.1. Tissue Sections
1. Use Teflon coated SuperFrostPlus slides (see Note 2).
2. Cut section of 2- to 5-µm thickness (see Note 10).
3. Change the microtome blade frequently and clean thoroughly with xylene after each block
to prevent crosscontamination between samples.
4. Incubate slides overnight at 50°C.
3.4.2. Cytospins
1. Centrifuge cells on slides.
2. Incubate slides for at least 30 min at 50°C.
3.5. Deparaffinization
1. Incubate slides for 10 min at 70°C.
2. Incubate slides for 10 min in fresh xylene (see Note 11).
3. Incubate slides for 2 min in fresh xylene.
4. Incubate 2 × 1 min in 100% ETOH.
5. Incubate 1 min in 90% ETOH.
6. Incubate 1 min in 70% ETOH.
7. Incubate 1 min in 50% ETOH.
8. Incubate 1 min in distilled water.
9. Dry Slides for up to 10 min at 37°C (on the thermocycler).
3.6. Permeabilization
The following permeabilization protocols are for optimized fixation conditions and
may vary with fixation conditions and tissues (see Notes 9 and 12).
3.6.1. By Protease
1. Incubate 15 min at 37°C (on the thermocycler) with 100 µL of Proteinase K solution.
2. Incubate 2 × 5 min in 0.1 M Tris-HCl, 0.1 M NaCl, pH 7.5.
3.6.2. By Target Retrieval
1. Put slides in a Coplin jar filled with 1 × Target Retrieval (Dako). Place Coplin jar in a
waterbath and incubate 35 min at 95°C.
2. Place Coplin jar outside the water bath and allow to cool for 20 min.
3. Incubate 2 × 5 min in 0.1 M Tris-HCl, 0.1 M NaCl, pH 7.5.
3.7. DNAse Treatment
1. Incubate in 100 µL of DNAse solution at 37°C for 12 h on the thermocycler. Cover slides
with coverslips.