Hi everyone,
I've recently started having some issues with free floating brain tissue sticking to itself after coming out of primary and secondary antibody incubation. Prior to antibody incubation, sections float and unfold readily. After incubation however, sections start to stick to themselves resulting in poor staining, shredding, and tearing of tissue.
These are 40micron coronal sections free floating in 24 well plates so they are incubated individually. This occurs for both fluorescent and colorimetric IHC.
For primary antibody incubation, I'm using 2% BSA with 2% serum in PBS with 0.25% Triton-X 100. Secondary is the same as primary without the addition of 2% serum.
Thank you,
Aidan
Hi Aidan,
We had a similar problem some time ago, which we believe was due to the different buffers we used. When slices were changed from a PBS-based to a Tris-based buffer (TBS for example), they (sometimes) became ultra-sticky. The solution was to always keep the same buffer. If we had to change, we would do that progressively, by first mixing the 2 buffers together before the switch.
If you always keep PBS in your incubation/washing steps, then I don't know where the problem comes from...Your protocol is very similar to ours (except we don't use BSA). Try checking the pH of your PBS, just to make sure that it's fine.
I hope you'll find a solution!
we have experience that problem before,but an overnight incubation in PBS really helped but we could not explain why.i hope you find a solution .cheers
Most important thing is rinsing. I use 3x10 min washing in PBS/TBS between every working step excluding between Blocking and primary antibody.
The storage time of your sections is essencial before you start with your immuno.
I do not store my sections longer then 4 weeks in the PBS in the fridge. If I will continue later or I need the material for other Immuno reactions then I deep freez them in a solution made out of Glycerol and 30% Succrose in PBS in equal parts at -20°C. Under this circumstances you can store your sections for years.
pH of the buffers: PBS pH 7.4, TBS pH 7,6-8; BSA should be prepared freshly. Don't forget to filter it before use. Good luck!
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