Expansion microscopy on lamina

Probes & Prep
1

Hi,
Does anyone have experience with expansion microscopy on lamina ?

I tried the protocol from Gambarotto, D.et al. (2018). Imaging cellular ultrastructures using expansion microscopy (U-ExM). Nature Methods . https://www.nature.com/articles/s41592-018-0238-1

Below is with methanol fixation. I tried without fixation and after immunostaining I couldn’t see anything.

Below and image of an expanded sample

image
image1093×1083 939 KB

But in reality it should look more like this (below a non expanded sample - not the same used for expansion)

image
image931×847 229 KB

Does anyone have any ideas on how to improve this ?

Thank you

2

I’m comparing the protocol (that I did) from

image
image1081×431 71.7 KB

with the one from
image .

I’s say that the major difference (other than the denaturation vs prot K) is that the Boyden one uses Acryloyl-X SE (AcX) to bind the proteins to the polymer matrix and U-ExM doesn’t.

I am unsure why the U-ExM method doesn’t use AcX but I wonder if this it the reason why my expanded sample looks poorly ?