Problem with epoxy silane (Random results in glass silanization). What are crucial points to look at?
The triethoxysilane group has to hydrolyze first in order to bond with a silanol group of glass surface. It seems that you are cleaning the slides very well. However, I think your temperature is too low and your time of exposure is too short. The hydrolysis of ethyoxysilane group is slow.
Secondly, I guess you are posting questions on this topic for almost a year without much success. In such cases, it is always good to go back and try a control experiment and think about a new approach. The control experiment is that you try just neat glass slides and see if your antibody sticks to glass or not. Glass itself is a good adsorber. Probably you might get similar results of &quot;fluorescence, no fluorescence and mild fluorescence&quot;. This will confirm that your silane binding procedure is doing nothing!
Second control test: Epoxy silane coated slides are commercially available. Why don't you try using them and see if your immobilization chemistry works on commercial slides or not. Schott makes commercial slides by this name: Nexterion® Slide E
Let me tell you how silanes are bonded onto silica. The reaction time is typically 10 hours under reflux conditions of toluene and other added solvents and catalysts! Sometimes a very very small amount of water is added to created polymer like bonding. There are at least several hundred papers on this topic. The surface area is silica is quite high so it is not a direct comparison with glass slides but still 3 min and room temperature is doing nothing!