[Histonet] re: phosphate buffered saline or Tris PBS ?

[Gayle Callis]

You can use either buffer for immunofluorescence work.  We buy Dulbeccos 
PBS from Sigma or make up TBS.  There are other PBS recipes that will work 
too.  Our TBS is 0.05M concentratin, commonly used for IFA and IHC work.

TBS (TRIS BUFFERED SALINE)
STOCK 10X TBS
TRIS Base (FW 121.41) 0.5M                        60.57 g
Sodium Chloride                                           89 - 90 g
Dissolve in 800 ml distilled water
Add 30 ml Hydrochloric Acid to adjust pH to 7.8, use magnetic stirrer with 
pH meter.
Adjust final volume to 1 liter and store in refrigerator.  If this becomes 
cloudy, toss and make new.  Good for 1 year.
0.05M TBS WORKING BUFFER
Dilute Stock 10X Buffer 1:10
100 ml Stock 10X TBS and 900 ml distilled water.
Final pH can be 7.6 - 7.8, final concentration of TBS is 0.05M in 0.9% 
sodium chloride.

TBS is a preferred buffer when you work with alkaline phosphatase 
immunohistochemical staining to eliminate phosphate ions that cause 
background staining with substrate/chromogen, but with IFA work, no real 
problems.

GFP can be rencered nonfluoresceing by certain things, solvents are one so 
we never use detergents.  You can find out more about GFP by going to 
Clontech website and accessing Living Colours Manual, free and in pfd format.

Personally and if you notice a reduction in GFP signal by using certain 
reagents, I would eliminate them or reduce the concentration.

t 03:54 AM 9/29/2005, you wrote:
>Hi,
>
>I'm currently using immunohistochemistry (free floating sections or 
>serially mounted sections) to map the rat brain.  We use viral injections 
>to express green fluorescent protein and use 1 or 2 other labels (i.e. Cy3 
>or AMCA) to colocalise receptors with GFP expression.
>
>I have been trying to improve our methods that we currently use. 
>Originally we used Tris PBS (0.01M, pH 7.4), I notice that other groups 
>use a variety of differences in these recipes, what advantages does Tris 
>PBS offer (if any) or would you recommend using PBS (either 0.1M or 0.01M) ?
>
>Secondly, when using triton x-100 (0.3%) has anyone noticed a reduction 
>with GFP signal compared to just using 50% ethanol ?
>
>And finally, we use cardiac perfusion to initially fix tissue followed by 
>2 hours post fixation with 4% phosphate buffered (0.1M, pH 7.4) formalin. 
>The antibody we use for mu opioid receptors (diasorin) - does anyone have 
>any experience with this antibody and this method have you done antigen 
>retrieval to improve the signal ?
>
>Many thanks
>
>Patrick
>
>----------------------
>Patrick Howorth,
>Dept of Physiology,
>University of Bristol,
>Bristol,
>BS8 1TD.
>+44 (0)117 33 17112
>patrick.howorth <@t> bristol.ac.uk
>
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Gayle Callis
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-6367
406 994-4303 (FAX)