Author Topic: about immunoTEM  (Read 8989 times)

0 Members and 1 Guest are viewing this topic.

Offline angela

  • SuperTech
  • ****
  • Posts: 48
about immunoTEM
« on: September 20, 2004, 01:54:37 PM »
Hi, I read the protocol for immunogold, and I have a question:
is it possible to do immunoTEM on tissues that have been fixed with gluteraldheyde and embedded in Epon or Araldite?
thank you

angela

about immunoTEM
« on: September 20, 2004, 01:54:37 PM »

Offline richard03

  • Administrator
  • GoldMember
  • *****
  • Posts: 1212
about immunoTEM
« Reply #1 on: September 21, 2004, 10:30:44 AM »
Hi Angela,

I would say it is possible but maybe difficult to reveal antigens since some (probably most) of antigens don't like gluteraldehyde fixation. For example, Dopamine receptors (D1, D2), etc. In this cases, people often use acrolein as an alternative.

However, I have read some literatures using antigen retrieval technique to increase staining intensity with gluteraldehyde fixed, Epon embedded ultrathin sections. You may want to search PubMed for more information.

The chances of getting positive results are low, but worth to try if it is critical data you need.

Richard

Offline immunoem

  • SuperTech
  • ****
  • Posts: 25
    • http://www.bath.ac.uk/~bssiwj/
about immunoTEM
« Reply #2 on: September 22, 2004, 03:04:24 AM »
Hi Angela,
yes, it is possible (see Cali Ingham's chapter in 'Experimental Neuroanatomy: A Practical Approach', ISBN 0-19-963325-8, for more details and a basic method). Unfortunatley not many antibodies work well on Epoxy resin sections, especially if they have been osmicated. You may like to check out Aldo Rustioni's web site as he and Kris Phend have developed an osmium-free embedding protocol to facilitate immunoTEM on epoxy resin sections (method at http://rustpc1.med.unc.edu/).
Good luck.
Ian

Offline Alexander Hilarov

  • Dr.
  • Newbie
  • *
  • Posts: 4
Re: about immunoTEM
« Reply #3 on: December 20, 2007, 05:00:48 PM »
Hi, I'm looking for a method alternative to immuno-gold revealing of antigents. I've heard that DAB (diaminobenzidine) being used in CLEM (correlative light electron microscopy). Briefly protocol was like this:
1) GFP-based time-lapse confocal microscopy
2) Fixation of the cells.
3) Immunoperoxidase labeling
4) Embedding in resin
5) Identification of antigen at EM level.

So, I wonder can I study paraffin embedded sections at LM level using routine immunohistochemical methods (DAB) and after that study the same specimen at EM levels?
Thank you.
Alexander

Offline richard03

  • Administrator
  • GoldMember
  • *****
  • Posts: 1212
Re: about immunoTEM
« Reply #4 on: December 26, 2007, 07:32:15 PM »
You can use DAB for ImmunoEM labeling, but DAB reaction products tend to diffuse so precise localization may not be determined or confused. So immunogold labeling has advantage over DAB.

Paraffin section can be used for TEM if you have no any other choices as the ultrastructure will be disrupted by paraffin embedding procedure.

Hi, I'm looking for a method alternative to immuno-gold revealing of antigents. I've heard that DAB (diaminobenzidine) being used in CLEM (correlative light electron microscopy). Briefly protocol was like this:
1) GFP-based time-lapse confocal microscopy
2) Fixation of the cells.
3) Immunoperoxidase labeling
4) Embedding in resin
5) Identification of antigen at EM level.

So, I wonder can I study paraffin embedded sections at LM level using routine immunohistochemical methods (DAB) and after that study the same specimen at EM levels?
Thank you.
Alexander

Re: about immunoTEM
« Reply #4 on: December 26, 2007, 07:32:15 PM »