Here’s a nice article from 2021: “Permeabilization-free en bloc immunohistochemistry for correlative microscopy” by Fulton and Briggman. A few thoughts:
1. The protocol the describd helps to overcome an impediment to correlative en bloc EM and IHC by enabling the ultrastructural preservation of brain tissue and antibody penetration into relatively thick tissue sections, without the use of permeabilizing agents. This could help to facilitate the use of pre-embedding IHC in ultrastructural analysis techniques such as 3D EM.
They replicate the finding that permeabilization dramatically decreases ultrastructural quality, so should be avoided if possible:
They argue that a key way they were able to accomplish antibody penetration without permeabilization was via the preservation of the extracellular space (ECS).
2. The brains sections they used are still quite thin relative to those that are practical in neuropathology, at 300 um – 1 mm. In neuropathology, human brains are somewhat frequently sectioned at 5 mm, but even that is challenging and requires expert skill.
One could try to use a device such as a compresstome to help with the sectioning process. Here’s a video showing how the compresstome works on mouse brains. But it seems difficult to scale this to human brain sizes.
(If one were to achieve such thin sections in a high-fidelity way, you could theoretically cryopreserve them with vitrification or near-vitrification procedures and therefore avoid fixation altogether. Although, avoiding fixatives would also make room temperature preservation not currently possible.)
3. Another possible mechanism for why their protocol worked, that the authors did not discuss as far as I could tell, is that tissue decomposition during the immersion fixation process — which is slower than perfusion fixation — may itself cause membrane permeabilization. With a long enough time period of decomposition, cell membrane breakdown is an inevitable event, so the question is really whether the immersion fixation was slow enough to allow it to occur. My guess is that it was a contributing factor.
This may also help to explain why some epitopes are more accessible (eg Homer) than others (eg PSD-95). If a protein is a part of stronger gel-like networks, this gel-like network will likely break down slower during the decomposition process, and therefore be more difficult for antibodies to access without permeabilization.
4. Do we even need immersion fixation for ECS preservation? They cite Cragg 1980 as an example of a study that achieved ECS preservation using perfusion. It’s still not entirely clear to me why perfusion doesn’t usually achieve ECS preservation, but it seems like it probably depends on the osmotic concentration of the perfusate. Cragg 1980 is 30+ years old now; it would be ideal if it could be replicated and the phenomenon understood better.