Hi,folks.
The thickness of my brain section is about 500um(the whole brain has been clearing for 1 week with 37℃,25V).And I did the immunostaining with the protocol provided by the authors(primary antibody:1d/washing:12h/ secondary antibody:1d/washing:12h). And follow the staining tips,eg 37℃ on a shaker.
But it was really depressed that the penetration depth of antibody was less than 100um.Even I increased the incubating time for both primary antibody and secondary antibody(3days) ,the depth was only about 100um.
Has anyone else experienced this? or any suggestion?
1 ·
Comments
I am also trying to image post mortem tissue with antibodies, and I didn't get my lucky shot yet. As stated in the article, a two days incubation of first antibody should suffice for imaging. The supplementary video included in the article shows a good overall staining in human brain. However, with an increase of incubation or with an increase of antibody concentration, I did not manage to reproduce these results.
Are there people here with working antibody stainings on post mortem human tissue? I do get the feeling that antibodies might be too large to pass the hydrogel barrier, because my RedDot stainings do work.
Hope somebody has suggestions here, cheers!
The authors said that 1mm for 3days per antibody and 14days for 5mm was what was needed for homogenous complete staining. Have you tried these extended times? We've been thinking about including higher concentrations of Triton X-100 and/or Saponin to see if it will assist us.
The time for diffusion should be T = (L^2)/(2*D), where T is time, L is the distance to diffuse across and D is the diffusion coefficient of the molecule in the system. A cancer immunotherapy paper by Jain & Baxter showed that in tumours (which are notoriously difficulty to penetrate) the time it takes a typical IgG macromolecule to diffuse through 1mm of tissue is ~54hrs and it should be noted that the relationship of diffusion time is proportional the to square of distance so the time becomes exponentially longer for deeper staining. Granted these are in vivo scenarios and exploiting vasculature to deliver the antibodies but this should give us some clues.
Another thread has suggested the use of Methnol/Freeze-thaw cycles/Proteinase K steps to increase penetration, which is currently being used in the SeeDB technique