[Histonet] RE: Immunogold question

[C.M. van der Loos]

   Hi Danielle and Robert,

   Indeed,  Robert  is  right  with  his response: the higher pH helps in
   reducing  background staining. It has something to do with the charges
   introduced  by the gold particles. At higher pH they are near neutral,
   at  lower  pH  the  gold particles are more negatively charged causing
   non-specific  binding  with  the  positively  charged  tissue elements
   (especially after aldehyde fixation). Changing the pH is certainly not
   the  only  thing  you  need  to  do  to  suppress background staining.
   Additives  like  BSA-c and Cold Water Fish Skin Gelatin applied in the
   right  incubation  step  also  contributes to a cleaner image. For the
   experts on this issue go to: [1]www.aurion.nl (English website).

   Cheers,

   Chris van der Loos, PhD
   Dept. of Pathology
   Academic Medical Center M2-230
   Meibergdreef 9
   NL-1105 AZ Amsterdam
   The Netherlands


   From: Danielle Crippen <dcrippen <@t> buckinstitute.org>
   To: histonet <@t> lists.utsouthwestern.edu
   Sent: Monday, January 29, 2007 10:27:46 AM
   Subject: [Histonet] Immunogold question
   Dear EM experts,
   I've been using the following protocol for immunogold labeling for the
   past  couple  years  with good success.  This morning one of our users
   has  inquired as to why the secondary is incubated at a higher pH than
   the primary.  I do not know the answer...do any of you???

References

   1. http://www.aurion.nl/