Dear all,
I would appreciate a lot if anyone could give me an advice how to resolve problem with non-speciffic labelling of immunofluorescently-lablelled mouse brain free floating sections, which occurs in red but not in green channel.
Briefly, I need to perform two-color staining of astrocytes (green channel) and glias (red channel). In order to establish the method, I tried to perform separate (one-color) staining of both markers. The staining of astrocytes works pretty well (image enclosed). On the other hand, samples stained for glias (staining was run in parallel in one experiment for both astrocytes and glias) gave signal not only for glias but I also see clusters of strongly fluorescent dots within the tissue. Although glias could be still identified, the final appearance of the image does not look very well (image enclosed).
The control samples performed with secondaries alone (without primaries) gave the same clusters of fluorescent dots in red channel. I performed titration of secondary antibody (1:500-1:2000) with the same result. Sections without secondaries give no such signal. It is apparent that there is problem in the secondary antibody itself.
Please, could someone suggest what could be a matter of the problem and how to resolve it?
Antibodies used:
- chicken anti-GFAP (SySy) -- goat anti-chicken Alexa Fluor 488 (Thermofisher)
- guinea-pig anti-IBA1 (SySy) -- goat anti-guinea-pig Alexa Fluor 555 (Thermofisher)
My protocol is:
Standard perfusion of the mouse with PBS-4% PFA, postfixation overnight, sectioning by vibratome (thickness of sections 40 um)
The labelling itself includes
- 3 washes with PBS 5 min
- permeabilization with 0,1% Triton/PBS 3x 15 min
- blocking with 10% filtered normal goat serum/0,1% Triton/PBS 60 min
- incubation with primaries in 0,1% Triton/PBS overnight at room temperature
-washing 3x15 min with 0,1% Triton/PBS
- incubation with secondaries in 0,1% Triton/PBS 2 h at room temperature
- washing 3x15 min with 0,1% Triton/PBS
Hi Petra
Try a spin of the secondary and take your Ab from the top (without disturbing the pellet), that usually helps when the antibody has precipitated
I generally find the red channel has a bit more background than others, although yours does look particularly faint. You could try staining in a different secondary in a different channel to see if it is an issue with the primary or secondary. Also make sure you are always protecting your samples from light once the secondary antibodies are on.
You could also play around with your primary antibody incubation times and concentrations, as well as your blocking solution.
A protocol that works well for me is:
- 3 washes with PBS 10 min
- blocking solution (3% BSA + 0.25% triton in PBS) for 1hr
- incubation with wako rabbit Iba1 (019-19741) at 1:1000 in blocking solution at 4°C for 72hr gently shaking on a orbital shaker.
-washing 3x10 min with PBS
- incubation with secondaries in blocking solution at 4°C for 24hr on a orbital shaker.
- washing 3x10 min with PBS
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