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,
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