Hello. I am doing an immunostaining of human iPSC adherent to a 384-well plate. Previosly I did a small-scale titration and optimized concentrations and incubation times of each step.
My problem is that I didn't realize it would be so difficult to scale it up to 384-well plate. The main reason for that is that we don't have automated pipets or multipipets in the lab, so I have to do all the washes by manually pipetting in/out of each well. Similarly, in previous experiments, I realized that using vacuum machine to aspirate leads to loss of a big proportion of my cells.
There are other reasons, but TL;DR: I am unable to perform fixing->permeabilization->blocking->staining of all 384 wells in one day (or even two). Humanly impossible. I've tried to do it in batches (2 rows at a time) but this means that I had to put the plate back in the incubator after staining them because the remaining rows have live cells in it. This greatly degrades the dyes apparently because I've just been in the imaging room and the signal is very poor.
The solution I am leaning right now is to change the protocol to look like the following:
Day 1: Fix and permeabilize cells. After permeabilization I wash them twice with PBS after second wash I leave them in PBS at 4ºC.
Day 2: Block, stain with primary antibody and wash 3x with PBS. Incubate with secondary overnight.
Day 3: Wash the second antibody and leave the cells in PBS at 4ºC
Day 4: Stain with DAPI, wash with PBS.
Day 5: Imaging
Do you think it will work? In principle if I wash them well and leave them at 4ºC, there should be no problem if the cells will stay in the fridge for one day before staining, since they are in PBS. Does anyone has a suggestion? Any help will be appreciated.
Hi Maksym,
Conducting this experiment in a 384 well plate without multichannel or automated pipettes will definitely not be a fun adventure. Our lab routinely does immunofluorescence but we typically do not run our analyses in greater than 96 well plates. Our protocol is similar to yours except, if we need to break the protocol overnight, we apply the block and then break the protocol. The other thing is we decant the block but do not do any washes before applying our primary antibody. This would save you quite a bit of hand strain (and time) if you can remove these washes. So ours is fix and permeabilize, wash, block, break overnight (4℃), primary, wash, secondary, wash, nuclear stain, wash, image. Hope this helps.
Leah
Leah Schneider Thank you for your answer! So you store the cells overnight at 4ºC in blocking solution? Our blocking solution is Normal Goat Serum at 5% in PBS... Maybe if it's OK to store it it Goat Serum + PBS, it could also be OK to store it in PBS alone? Thank you again.
We use 3% BSA in PBS for our block. We have never tried PBS alone, so I can't speak to that.
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