[Histonet] Acetone freeze substitution vs fixation, and other Cryo questions

[Evan Chiswick]

Hello Histonet members,

I am fairly new to the world of histology, but I have found this listserve to be very helpful.

I have a few questions which will be followed by a general description of my project/protocol, in case that is helpful for answering.


Question: I have read here and elsewhere of acetone used as a fixative after first drying the section, but other fixatives are used when acetone is used for freeze substitution(e.g. http://jcb.rupress.org/content/jcb/4/5/593.full.pdf). Does freeze substitution with cold acetone also fix the section? I want to avoid air drying the section as I have the impression that will distort the morphology.

What happens to freeze-substituted sections when I subsequently immerse then in staining buffer (.e.g. PBS w/BSA & Triton-x). Would that reverse the substitution/fixation?

If I want to subsequently store my stained slides for future use/reference, how should I do that? How should freeze substituted slides be stored (pre-staining)



Brief description of project.

Use highly multiplexed immunofluorescent staining of uterine sections to measure cellular composition, protease activity (amenable to acetone/frozen sections), as well as various other molecular features. Through computational methods, these features will be modeled in conjunction with the spatial and morphological features of the section. Hence, I want a tissue preservation method that preserves enzymatic activity (freezing) and morphological detail (freeze substitution/no air drying??). I am under the impression that air-drying is necessary to ensure attachment of sections to slide. Would the cryojane tape-transfer technique get me around the need for air drying?



Brief outline of protocol

Snap freeze tissue blocks in isopentane chilled by liquid nitrogen.

Section 4um with cryojane, freeze substitute with cold acetone, and store at -80 or liquid nitrogen.

Block sections (PBS/BSA Triton-x100, or something similar).

Stain with panel of antibodies with unique linkers attached. Wash.

Light PFA fixation (~10min) to crosslink antibodies.

Iteratively image 3-4 antigens at a time, with cyclic detection incubations, image, dehybridize detection, repeat).

Store slide for future use (e.g. additional immunostains, or H&E) (how to store?)



Thank you for your time.

Sincerely,

~Evan

Evan L. Chiswick
Postdoctoral Research Associate
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Department of Biological Engineering
Massachusetts Institute of Technology
Cambridge, MA