Hello everyone,
I am gearing up to prepare my first ImmunoTEM samples via a pre-embedding protocol. The samples are isolated mouse sperm. I searched the literature and I am gathering bits and pieces from different papers and book chapters to form my final protocol (so many methods sections are grossly incomplete).
I understand why the fixative is excluded before antibody incubation, but I did run across many papers where a brief fixation with paraformaldehyde (4% mostly, but one paper used 8%!) at the very beginning of the procedure was employed. The silver enhancement kit that I am considering also listed an optional fixation with glutaraldehyde (.05-1%) alone at the start of the procedure.
Should I include a brief fixation at the start? Do I permeabilize the cells before this or only before I start antibody incubations?
I've written up a protocol and could share it via private message if anyone is willing to look it over.
Any help is greatly appreciated! Thanks!
So much depends on the properties and location of the antigen. Glutaraldehyde ruins most antigens so they can't be recognized by the antibody (much faster than formaldehyde does), but gives better preservation of structure at the EM level. If you can demembranate your sperm and the antigen is still insoluble, then it may be easy to incubate demembranated sperm with antibodies (primary and secondary) without any fixation, then fix and embed as you would for any TEM specimen. You can check by running quick tests with a fluorescent secondary, or even with gold secondaries by negative stain EM on whole mounts, much faster than embedding and sectioning. See attached pub for whole mount method details.
Hi David!
My collaborator has already done some immunofluorescence/STORM work with the same antigen, so I am guessing it is accessible. I am planning on purchasing 1.4nm particles, so that should help. We will try the negative staining just to be sure, though. Thank you for the paper!
I ditto all of David's comments! Doing the AB binding first (incl. secondary with at least 5 nm gold, so you can see it), would allow you to do a TEM specimen prep that focused on structure preservation (instead of epitope preservation). ... That your collaborator has already gotten FLM to work, tells you how to make the primary AB work (usually includes fixation (e.g. paraform.) and membrane-permealization), but it doesn't mean much for the structure preservation - in fact, FLM specimen look usually horrible in TEM (only in FLM they just see "their structure of interested" and not the rest of the structures). ... Not sure why you would want to go in with gold that you can't see just to have to add Ag-enhancement steps? Good luck.
One of the specialists in this technique is one of my former coworkers, Michael Hoppert., University of Goettingen/Germany, Institute of Microbiology and Genetics. He is also a member of Researchgate: Just ask him!
Best regards,
Frank Mayer
- Badges
- Science topic
- Similar topics
- Medicine
- Disease
- Disease Models
- Animal Models
- Rodent Models
- Mouse Models