Methods and Techniques Discussion > Immunofluorescence (IF)

Non-specific speckles on IF of boar testis tissue


Hi there,

I am getting some non-specific speckling on my recent IF slides and have not been able to fix the issue. I can tell they are non-specific because even when I scroll off my tissue to the surrounding slide, there are speckles. The neg control is 100% clear. No speckles. Otherwise, I do get signal. The problem is it looks so messy and the speckles are very bright compared to the actual signal. Here is my protocol:

Depara with xylene
antigen retrieval with 1 X Na Citrate
Cool 2 hr
Rinse in TBS
Block for 1 h in 10% normal serum (goat; diluted in TBST (0.05%))
Primary 1 hr ovn (1:50; in block)
Rinse 5 min in TBST (3x)
Secondary (Alexa Flour 555, brand new, diluted in block) for 1 hr at RT (various concentrations I am trying, all yield speckles)
Rinse 5 min in TBST (3x)
10 min 0.3% sudan black B (to eliminate autofluorescence)
Rinse 5 min in TBST (3x)
Mount and seal

Here is a link to the secondary info:
If you have any suggestions, I would love to hear them!!

Some time ago I saw similar effects. It was funny, because it wasn't there, when we did a drying step of the tissueslide at 60C oven for a few hours. At last I thought it had something to do with the adhesive slides' surface. - but no real solution of the problem.

I figured it out!!!! Antibody aggregates. If I spin down for 10 min (13000 x g) at 4 degrees, the speckles are gone! Thank goodness.

Congratulations!!  :)


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