Hi everyone, I have been trying for weeks to get this simple stain to work, but no matter what I do, nothing works, so now I have come to Reddit. I am performing immunofluorescence on 40-µm brain sections (PFA-fixed and sectioned on cryotome), staining for Neurofilament Light chain in the optic tract. However, I am encountering a problem. The edges of my tissue look great under the confocal, but the signal drops off around a quarter of the way in, leaving a large signal void in the middle of my tissue. I've tested the following modifications to my protocol to no avail:
- Increase TritonX concentration for permeabilization from 0.1% -> 0.5%
- Longer primary antibody incubation at room temperature: 1 day -> 2 days
- 3-day primary antibody incubation at 4 ºC
- 24-hour primary antibody incubation at 37 ºC
- Higher primary antibody concentrations, as much as 1:500
- Higher antibody dilutions (serial), up to 1:160,000 (signal is almost entirely gone at 160,000)
I've validated that my secondary antibodies work. Used them on a different primary for a separate stain, and they penetrated all the way through. I'm on the verge of giving up, but if anyone has any recommendations, I'd love to hear them. Thanks!