TRIS-Buffered Glycerol, pH 7.2

Recipe: Tris-Buffered Glycerol (TBG), 5% n-Propyl Gallate (nPG), pH 7.2

To make 10 ml TBG 7.2:
● Add 0.5 g nPG to 1 ml 2M Tris, pH 7.2*
● Add nPG/Tris to 9 ml glycerol.
● Mix well, aliquot and store at -20º C

For 50 ml TBG 7.2:

● Add 2.5 g nPG to 5 ml 2M Tris, pH 7.2*

● Add nPG/Tris to 45 ml glycerol.

● Mix well, aliquot and store at -20º C

Note:

1. 2M Tris = 1.6 g Tris HCl + 0.134 Tris Base in DI (blue-cap or better). Note that a calomel electrode is needed to accurately measure Tris pH
2. nPG goes readily into solution at about 60ºC. Use a water bath heated to 65 - 70ºC to allow time to vortex nPG solution and add to glycerol without precipitating out of solution (and have glycerol measured and ready...)
3. 500 µl aliquots are enough to coverslip 6 to 8 slides depending on coverslip size, and yield at best 20 aliquots from 10 ml. Scintillation vials and 50 ml centrifuge tubes work well for making larger volumes
4. nPG is an antifade agent that slows bleaching of cyanine and alexa dyes, but can be omitted if desired.
5. Recipe was adapted from Confocal Microscopy for Biologists (Hibbs, A.R., 2004; Springer).

Results & Rationale
Originally tested 8/16/2012 as a means of limiting the excitability of Fluorogold (FG) by wavelengths longer than UV, or at least < 488 nm to keep it out of the green (and red channels). 

Two TBG mounting media at pH 7.2 and 7.6 were compared to the normal bicarbonate (KHCO3)-buffered glycerol (BBG, pH 8.6), and tested on FG-injected as well as green- or red-labeled peptide IHC.

Both TBG media both reduce the number of FG-labeled neurons visible in the green and red channels compared to BBG without attenuating the FG signal or interfering with normal peptide IHC. However, TBG, 7.2 was clearly more effective, eliminating all neuronal labeling in both channels. At the injection site, FG labeling was clearly visible in the green and red channels when mounted w/either BBG or TBG 7.6, but faintly visible in the green channel only w/TBG 7.2.