1. Drop 10 μl of cell suspension onto a slide* and wait till the surface becomes granule-like, i.e. ethanol meniscus occurred on the top of the cells, (10–15 sec)
2. Drop 18–22 μl of fixative (1:1, 2:1, 3:1 or 5:1 ethanol:acetic acid)** and wait till the surface becomes granule-like and the layer of fixative becomes thin (25–35 sec)
3. Put the slides upside down under the steam from a water bath at 55°C (10–15 cm from water surface of the water bath) for 3–5 sec
4. For double “SteamDrop”, repeat step 2 but with less volume (3–6 μl) of fixative and higher concentration of acetic acid. Perform Step 3 for 1 sec only.
5. Immediately dry slides with air flow (e.g. a tabletop fan).
Note
*-for preparation of large size chromosomes it is useful to coat slides with APES (3-aminopropyl-triethoxy-silane) to prevent a chromosome partial detachment. APES coating of slides: 1.5% APES in 100% acetone for 30 sec, twice wash in distilled water and dry for 1 h at 37°C.
**– the protocol allows an easy correction of enzyme treatment results - check the level of tissue enzymatic digestion in the first chromosome preparation slide under microscope, if tissue is underdigested use a high proportion of acetic acid in fixative (1:1 or 2:1); if tissue is overdigested use a low proportion of acetic acid in fixative (5:1 or 10:1).
2. Drop 18–22 μl of fixative (1:1, 2:1, 3:1 or 5:1 ethanol:acetic acid)** and wait till the surface becomes granule-like and the layer of fixative becomes thin (25–35 sec)
3. Put the slides upside down under the steam from a water bath at 55°C (10–15 cm from water surface of the water bath) for 3–5 sec
4. For double “SteamDrop”, repeat step 2 but with less volume (3–6 μl) of fixative and higher concentration of acetic acid. Perform Step 3 for 1 sec only.
5. Immediately dry slides with air flow (e.g. a tabletop fan).
Note
*-for preparation of large size chromosomes it is useful to coat slides with APES (3-aminopropyl-triethoxy-silane) to prevent a chromosome partial detachment. APES coating of slides: 1.5% APES in 100% acetone for 30 sec, twice wash in distilled water and dry for 1 h at 37°C.
**– the protocol allows an easy correction of enzyme treatment results - check the level of tissue enzymatic digestion in the first chromosome preparation slide under microscope, if tissue is underdigested use a high proportion of acetic acid in fixative (1:1 or 2:1); if tissue is overdigested use a low proportion of acetic acid in fixative (5:1 or 10:1).
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