Dear colleagues,

We are trying to do FISH on mice PDEC metaphase spreads. We use homemade biotin labelled probes, synthetized using a nick translation kit.

For hybridization, initially we were using leftovers of an hybridization buffer that came with a commercial probe (https://www.abnova.com/en-global/product/detail/FC0055) to dilute our homemade probe. It worked nicely (see attached image 1).

When we ran out of this hybridization buffer, we tried to make our own hybridization buffer:

- 600µL Formamide (final concentration 60%)

- 0,1 g Dextran sulfate sodium salt (final concentration 10%)

- 100 µL 20x SSC (final concentration 2x SSC)

- 200 µL H2O

Since using the homemade buffer, the chromosomal spreads look shrunken, and the interphase nuclei disrupted (see attached image 2). We bought another commercial hybridization buffer (https://www.sigmaaldrich.com/ES/es/product/sigma/h7782) but the problem persisted. These two last buffers, homemade and Sigma Aldrich, are much more dense and less bubbly than the Abnova one that worked nicely.

After preparing the metaphase spreads, we check them on a phase contrast microscope and they look fine, so we know they are messed during hybridization or posterior steps.

The protocol we are following for FISH, for all tested hybridization buffers, is:

Dehydration of metaphase spreads:

Wash briefly the slides with 2x SSC.

Dehydrate the slides in ascending dilution series of EtOH: 70, 85 and 100% for 2 min each.

Allow them to air-dry in the bench.

Prepare the hybridization solution, 25 µL per slide:

- 23,75 µL Hybridization Buffer

- 1 µL Probe

- 0,25 µL Salmon Sperm DNA 10 mg/mL (final concentration 100 µg/mL)

Add 25 µL of hybridization solution on each slide.

Place a coverslip on top of the slide, so the hybridization solution spreads.

Place the slides on a humid hybridizer:

- For denaturation of cell’s DNA and probe: incubate at 85ºC for 5 min.

- For hybridization: incubate at 37ºC overnight.

Post-hybridization washings:

Pre-heat bath at 42ºC.

Remove coverslips.

Wash in 0,4x SSC for 2 min at 42ºC.

Wash in 2x SSC with 0,1% Tween-20 for 2 min at 42ºC.

If using biotinylated probe:

Add 200 µL of blocking solution 3% BSA 0,1% Tween20 4x SSC. Cover with parafilm and incubate at RT for 1 h.

Remove coverslips, let dry the slides shortly. Add 30 µL of Streptavidin solution 2 µg/mL Streptavidin in 1% BSA 0,1% Tween20 4x SSC. Cover with parafilm and incubate at RT for 1 to 2 hours.

Wash 3x with 0,1% Tween20 4x SSC for 8 min per wash at 42ºC.

DAPI counterstain and mounting:

Apply 5 µL of DAPI 1 mg/mL.

Incubate 10 min at RT.

Wash with 2x SSC for 5 min.

Add a drop of mounting media.

Place a glass coverslip and press gently to remove fluid excess.

Please, does anyone know why some hybridization buffers mess the metaphase spreads? Thank you in advance.