Re: [Histonet] cacodylate vs PBS

From:Sharon Cooperman

Hi.  Actually, I've tried both and my best IHC and tissue quality 
seems to be with PBS, but I think that may be because I started with 
NaCacodylate and switched to PBS later and my techniques got better. 
I'll try Cacodylate for the antibodies I use against membrane 
proteins to see if that works better than PBS.


>Hi Sharon:
>    Rather than believe other's opinions (unless they have references 
>to back them up) the only real way to answer the question is to 'do 
>the experiment' yourself. The best "ammunition" is real-world 
>results in your lab with your antigens. Sodium cacodylate has an 
>advantage as a buffer for EM fixation in that you can dissolve 
>calcium ions in it (for improved membrane fixation). Calcium 
>phosphate, on the other hand, is very insoluble (this fact does not 
>seem to deter some folks from putting Ca ions in their 
>phosphate-buffered fix, they happily filter out the precipitate and 
>continue on their merry way). Cacolylate has arsenic in it and 
>should be disposed of with much greater care than phosphate buffers. 
>Cacodylate does seem to last longer before molds begin to grow in it.
>    After enduring the rigors of paraffin processing,  I would be 
>surprised if the choice of buffer made much difference, but it 
>might, depending on the antigen in question.
>Sharon Cooperman wrote:
>>Hi, histonetters.  There is a debate in my lab regarding which 
>>buffer is best for IHC on FFPE tissue.  I use 0.1M PBS made up the 
>>usual way (Sorensen's?)  Other people in my lab use NaCacodylate. 
>>I have been told by various people that NaCacodylate is used for 
>>some EM purposes and doesn't preserve antigenicity or isn't the 
>>best choice for IHC for some other reason.  Does anyone have info 
>>or an opinion on this to either set me straight or provide me with 
>>ammunition for the next round of arguing?
>Geoff McAuliffe, Ph.D.
>Neuroscience and Cell Biology
>Robert Wood Johnson Medical School
>675 Hoes Lane, Piscataway, NJ 08854
>voice: (732)-235-4583; fax: -4029

Sharon Cooperman       	     
NIH, NICHD, CBMB                     301.435-7735
Building 18T, room 101               301.402-0078 fax
Bethesda, MD 20892

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