TABLE 1

Steps and methods involved in optimization of FISH

ProcessMethodSource or reference
Fixation2% PFA for 30 min at RT31, 32
2–4% PFA for 1–24 h33
5–10% Formalin overnight34
4% PFA for 12–24 h at 4°CThis study
StorageCollect and resuspend cells/pellets in molecular-grade 100% EtOH and store at −80°C
Permeabilization5 mg ml−1 lysozyme for 30 min at RT31, 32
96% EtOH29
7% Tween 20
0.1% DEPC for 12 min at RT or 0.5% SDS for 10–15 min31
5 mg ml−1 lysozyme 30 min at RTThis study
1% SDS for 1 h at RT
Quenching0.1% DEPC for 12 min at RT31, 32
1–3% H2O2 for 60 min at RTKit
0.1% DEPC for 12 min at RT29
0.01% H2O2 for 10 min at RTThis study
Probe concn250 ng μl−1 for 24 h31, 32
25 ng μl−1 for 2.5 h29
50 ng μl−133
Probe concn vs cell concnThis study
Fluorophore concn0.25–0.5 μg ml−1 for 5 min at RT31, 32
1:5029
1:100 for 5–10 min at RTKit
1:100 for 30 min at RTThis study