[Histonet] Phosphate buffered saline (PBS) with and without CaandMg

[Gayle Callis]

We add sucrose to Mg and Ca free Dulbeccos PBS for cryoprotection.  This is
the same DPBS we use for immunostaining but without the sucrose!  It works
well, and tissues are fine. When immunostaining cells cultured on multiwell
chamber slides and destined for immunostaining, we use the same DPBS that
we use for frozen sections.   

  

At 03:08 PM 1/23/2004 -0800, you wrote:
>I am using PBS for making paraformaldehyde fix and also for 30% sucrose for
>cryoprotecting tissues for both immunostaining and for conventional
>histochemistry. I have the idea that the calcium and magnesium is required
>when you are working with tissues as opposed to cells, since the Ca and Mg
>are important for cell adhesion. With cell culture you want the cells to
>stay separate so that's when to use the Ca/Mg free PBS. Am I correct?
>
>For the sucrose, which is used on fixed tissue, would it still be necessary
>to use PBS with Ca/Mg, since now the tissues are fixed they should not
>dissociate if Ca/Mg free PBS?
>
>-----Original Message-----
>From: Gayle Callis [mailto:gcallis <@t> montana.edu]
>Sent: Friday, January 23, 2004 2:41 PM
>To: cmather <@t> origentherapeutics.com
>Subject: Re: [Histonet] Phosphate buffered saline (PBS) with and without
>Ca andMg
>
>
>I presume you are using the PBS for immunostaining?  You did not say what
>application was needed. We use Dulbeccos PBS without Ca and Mg for
>immunostaining of all kinds.  For Beta Galactosidase or Beta Gal stained
>tissue sections/whole lungs, the Dulbeccos has Mg added, probably to aid
>enzyme reaction and the method has stringent directions.
>
>People who do cell cultures and some other molecular biology applications
>in our department sometimes use Dulbeccos PBS with the Ca and Mg added as
>essential nutrients? during cell growth. Not being a cell culture type, I
>may have not answered this completely.
>
>I think for most immunostaining purposes, rinses, etc, plain PBS is the
>standard reagent unless otherwise specified for some special enzyme or
>other method protocol. If a method calls for Mg and Ca, then you need it.
>
>At 02:21 PM 1/23/2004 -0800, you wrote:
>>What are the essential differences in the uses of PBS with and without
>>calcium and magnesium. Why would you use one in favor of the other?
>>
>>Christine.
>>
>>biotech employee
>>
>>
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>>
>Gayle Callis
>MT,HT,HTL(ASCP)
>Research Histopathology Supervisor
>Veterinary Molecular Biology
>Montana State University - Bozeman
>PO Box 173610
>Bozeman MT 59717-3610
>406 994-6367 (lab with voice mail)
>406 994-4303 (FAX)
>
>
>
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>
>
Gayle Callis
MT,HT,HTL(ASCP)
Research Histopathology Supervisor
Veterinary Molecular Biology 
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-6367 (lab with voice mail)
406 994-4303 (FAX)