Hi everyone,

I have cells on top of a layer of Matrigel (60 uL of matrigel in a 96-well plate) that i'm trying fix for immunofluorescence. I've tried a PFA fix and a methanol fix, but in both techniques, a majority of cells would wash off by the end.

Thanks to this community, i read up on using 1% Glutaraldehyde followed by a 1% NaBH4 quench. I tried this, and it looks like it works. A lot of my cells are still there by the end. Unfortunately, the NaBH4 step also produces a whole lot of bubbles. Even worse, these bubbles seep into to the matrigel, and since I'm using a 96-well plate, these bubbles can make a large portion (or all) of the well unreadable.

I've tried a couple things:

1. Using less NaBH4 to quench (have tried 1%, 0.5%, and 0.1%)

2. Dissolving the NaBH4 in PBS and water

3. Not moving the plate at all after adding NaBH4

But the bubbles are still there. Tried contacting Corning, but they said pipette from the middle of the solution not the top (duh), but even the pipetting action itself introduces bubbles into the pipette tip.

Any help? How do people get rid of the bubbles? Everywhere I read says to use the NaBH4 solution fresh - but this is when it has the most bubbles. Am I supposed to wait a little bit? The fact I cant find a way to fix/stain these cells is driving me nuts. Please help!