We are conducting our first experiment with immunofluorescence imaging.

We plan to measure expression and coexpression of two proteins in lymph nodes.

We use Olympus scanner VS-120 with custom made filter cube (parameters attach).

Our scanner is set for DAPI/FITC/Cy3/Cy5 fluorochromes

ProteinA (rabbit antibody) is labelled with Alexa 647 dye -red colour, channel for Cy5.

ProteinB (mouse antibody) is labelled with Alexa 488 dye – green colour, channel for FITC .

We have tested antibodies in IHC stains.

As you can see Alexa 488 signal seems to look very similar to that on IHC (large parts of cytoplasm stained).

Alexa 647 seems to work too ( there dot-like cytoplasm stain) but there is also seen signal that is exactly the same as signal from Alexa 488.

I have scan slides in third channel that have extension and emission values between that for Alexa 488 and Alexa 647 (yellow colour, channel for Cy3). I don’t see such signal there.

W use following staining protocol:

1. Block: PBS + 5%NGS +1%BSA+ 0,05% Tween 20 – 1h

2. Wash: PBS+ 0,05% Tween 20- 10min x 3

3. Primary antibody diluted in PBS + 1%BSA+ 2%FBS+ 0,1% Triton X-100 – 12 h

4. Wash: PBS+ 0,05% Tween 20- 10min x 3

5. Secondary antibody diluted in PBS+ 1%BSA+ 2%FBS+ 0,1% Triton X-100 – 1h

6. Wash: PBS+ 0,05% Tween 20- 10min x 3

Antibodies:

Primary: Rabbit anti-A

Mouse anti-B

Secondary: Goat anti-mous Alexa-488

Goat anti rabbit Alexa-647

Could you help me explain this phenomenon?

I’m guessing it have something to do with our labelling protocol or maybe we choose wrong settings for our cube/filters?

I would be gladly hear any advices.