My lab performs immunohistochemistry and uses non fat dry milk powder dissolved in PBT-Azide (phosphate buffered saline with Triton-X-100 and sodium azide) for the blocking buffer. We recently changed our PBS recipe as it wasn't properly buffering solutions before. Now it works as a buffer, but ever since the switch we have observed "clumps" forming in the milk when we leave it overnight.

The recipe in which the clumps form contain 0.1M sodium phosphate dibasic anhydrous, 0.1M sodium phosphate dibasic, 0.13M NaCl, and 0.25% Triton-X-100 at pH 7.4. I have tried playing with the Triton-X-100 concentration, using unopened bottles of chemicals and NFDM to ensure no contamination, adjusting the pH, and wrapping our well plates in parafilm. The only thing that has helped is the parafilm, but only moderately, as we are still observing clumping. The clumps are more severe in solutions with Triton-X-100, however we can still see that it is happening more in any of our PBS solutions made with the new recipe compared to how it was with our previous recipe. Our main suspicions were either contamination, or the acidity of the solution. We have ruled out contamination, and the acidity does not seem likely as solutions with a lower pH have shown less clumping. The addition of non fat dry milk powder to a solution changes the pH, however depending on the pH of the original solution, it raises or lowers it.

Any ideas for troubleshooting would be greatly appreciated! Thanks in advance.