[Histonet] sodium citrate antigen retrieval protocol

[Rene J Buesa]

Citrate buffer pH6 recipe:
  Sol. A: 21.0 g of citric acid monohydrate + 1 liter of dist. water
  Sol. B: 29.41 g of sodium citrate dihydrated + 1 litre of dist. water
   
  To prepare 750 mL of citrate buffer:
  Mix 13.5 mL of Sol.A + 61.5 mL of Sol. B + distilled water UP TO 750 mL.
  Adjust pHto 6 (with NaOH if pH<6 or with HCl if pH>6).
   
  My question: why on free floating brain sections? Is it necessary that way? Why not on sections adhered to a glass slide? [Just my "protocolar" ignorance!].
  A free floating section will undergo the wrinkling you refer to.
  Could you adhere the sections to a platinum loop and process them that way (as it was customary about 50 years ago for chick embryos?). Just a thought!
  René J.

N Fournier <nfournier <@t> sasktel.net> wrote:
  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


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