[Histonet] sodium citrate antigen retrieval protocol

[N Fournier]

Dear Colleagues,

I need some assistance regarding how to do  heat-induced antigen retrieval for on free-floating brain sections.  I am using the protocol suggested by Jiao et al., (1999) in which they used 10 mM sodium citrate pH 8.5-9.0 for 30 min at 80 degree C.    The recipe I used was 2.94 g  tri-sodium citrate (dihydrate) in 1000 ml dH2O and pH the solution using 5 N NaOH.  I preheated culture wells that contained sodium citrate at 80-82 degree C for about an hour or so before the antigen retrieval step was needed.  We use a isotemp Fisher water bath for heating (we do not have access to a microwave).  I placed netwells containing the sections into the preheated culture dish (6 well) and returned them to the heat bath. .  

When i used this protocol and evaluated the sections at the end of an immunostaining for synaptophysin or Prox-1,  I found it produced extensive wrinkling and damage to the tissue.   Any suggestions.  Should I be using citric acid in the recipe for sodium citrate buffer?  If so, does anyone have a recipe for the protocol and importantly the amounts of citrate acid and sodium citrate needed to produce a 10 mM solution.  

I appreciate your help,

Neil