TITLE: Protocol to immunostain Gastruloids (LSCB, EPFL)
AUTHORS: Stefano Vianello, Mehmet Girgin, Giuliana Rossi, Matthias Lutolf

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Standard protocol used for immunostaining Gastruloids (aggregates of mouse embryonic stem cells) in the Lutolf Lab, EPFL.</div><div class = "text-block">For a published protocol on how to generate Gastruloids, see: </div></div>

[STEPS]
?. [Fixing and primary antibody stain (D1)]
Harvesting Gastruloids:
?. [Fixing and primary antibody stain (D1)]
Using a cut P1000 tip to avoid accidental damage (cfr. "Guidelines"), collect Gastruloids from each well of the 96-well plate by placing the tip straight down to the bottom of each well, and aspirating up. Move to the next well until full tip capacity is reached.
To increase reproducibility of analysis, do not collect Gastruloids from the outer wells of the plate (all 36 border wells). These tend to develop differently from all other Gastruloids, probably due to evaporation of the medium. We only collect Gastruloids from the inner 60 wells of the plate.
?. [Fixing and primary antibody stain (D1)]
Deposit collected Gastruloids in a 15mL centrifuge tube as you go
?. [Fixing and primary antibody stain (D1)]
When collected all Gastruloids needed, wait for them to sediment to the bottom of the tube, and remove as much medium as possible (vacuum pump+glass pipette)
?. [Fixing and primary antibody stain (D1)]
Dilute away left-over traces of medium by gently resuspending the Gastruloids in ~5mL PBS-/-
?. [Fixing and primary antibody stain (D1)]
Fixing (4%PFA):
?. [Fixing and primary antibody stain (D1)]
Remove the serum-containing medium (in our case, PBSFT) from the 6-well plate (can keep and recycle to coat tips; cfr. "Guidelines") and replace with 2mL 4%PFA
PFA is toxic. Operate under a fume hood and wear appropriate hand, body, and eye protection.
?. [Fixing and primary antibody stain (D1)]
Collect all Gastruloids from the tube, wait for them to sink at the bottom of the tip, and touch the surface of the PFA vertically to transfer them with minimal liquid transfer (cfr. "Guidelines")
Coat a cut P1000 tips in PBS+FT to avoi Gastruloids sticking to it!
48h Gastruloids are particularly tricky to work with: they will stick and clump together, and they are almot invisible to the naked eye. To facilitate working with this timepoint, just push the whole contents of the tip into the next wash, without waiting for them to sink to the bottom of the tip, and without minding the carry over of medium from the previous wash. If possible, work with an excess of Gastruloids to compensate for loss during staining and washes.
?. [Fixing and primary antibody stain (D1)]
Cover with aluminium foil to reduce fluorescence loss (when using reporter lines)
?. [Fixing and primary antibody stain (D1)]
Incubate at  , with or without orbital shaker
4 °C
?. [Fixing and primary antibody stain (D1)]
Washes (to remove PFA):
?. [Fixing and primary antibody stain (D1)]
Fill wells of a 6-well plate with 4mL PBS-/- and transfer fixed Gastruloids using the same technique as above
?. [Fixing and primary antibody stain (D1)]
Cover with aluminium foil and wait  min
?. [Fixing and primary antibody stain (D1)]
repeat for two more washes PAUSE POINT: you can leave Gastruloids in the last PBS wash, for months,  , protected from light
If short on time, it is not necessary to wait 10 minutes for each wash. Gastruloids can be transferred serially across three PBS-filled wells, and left 10 minutes only in the last one.
4 °C
?. [Fixing and primary antibody stain (D1)]
Blocking:
?. [Fixing and primary antibody stain (D1)]
Transfer Gastruloids to a well filled with 2mL PBS+FT
?. [Fixing and primary antibody stain (D1)]
Block for  h,  , with or without shaker
0 Room temperature
?. [Fixing and primary antibody stain (D1)]
Primary antibody staining:
?. [Fixing and primary antibody stain (D1)]
For every antibody combination and sample condition you want to stain, prepare a 1.5mL Eppendorf tube with 500uL of the appropriate antibodies in PBSFT. Include DAPI 1:500.
?. [Fixing and primary antibody stain (D1)]
Transfer each antibody solution in a separate well of a 48 well plate, and transfer equal number of Gastruloids to each well
?. [Fixing and primary antibody stain (D1)]
Cover in aluminium and incubate  ,  , on a orbital shaker
4 °C
?. [Secondary antibody stain (D2)]
Washes (to remove 1ry Ab):
?. [Secondary antibody stain (D2)]
Wash Gastruloids by transferring them to a well of a 6-well plate filled with 3mL PBS+FT
Previous versions of this protocol call for PBS-/- instead of PBS-FT. There is a much higher increase of Gastruloids still sticking to the bottom of the wells in this case however. PBS-FT seems to be the safest option.
You will need a separate PBS-FT well for each different sample condition, do not mix all Gastruloids into the same well
?. [Secondary antibody stain (D2)]
Wait for  min
?. [Secondary antibody stain (D2)]
Repeat for two more washes   (total:  h)
Again, if short on time, it is not necessary to wait 20 minutes for each wash. Gastruloids can be transferred serially across three PBSFT-filled wells, and left 20 minutes only in the last one.
?. [Secondary antibody stain (D2)]
Secondary antibody staining:
?. [Secondary antibody stain (D2)]
For every antibody combination and sample condition stained, prepare 500uL of the appropriate secondary antibodies in PBSFT. Include DAPI 1:500 at this step too.
?. [Secondary antibody stain (D2)]
Transfer each antibody solution in a separate well of a 48 well plate, and transfer each Gastruloid set to its appropriate secondary solution
?. [Secondary antibody stain (D2)]
Cover in aluminium and incubate  ,  , on a orbital shaker
4 °C
?. [Mounting (D3)]
Washes (to remove 2ry Ab):
?. [Mounting (D3)]
Wash Gastruloids by transferring them to a well of a 6-well plate filled with 3mL PBS+FT
Previous versions of this protocol call for PBS-/- instead of PBS-FT. There is a much higher increase of Gastruloids still sticking to the bottom of the wells in this case however. PBS-FT seems to be the safest option.
You will need a separate PBS-FT well for each different sample condition, do not mix all Gastruloids into the same well
?. [Mounting (D3)]
Wait for  min
?. [Mounting (D3)]
Repeat for two more washes   (total:  h)
Again, if short on time, it is not necessary to wait 20 minutes for each wash. Gastruloids can be transferred serially across three PBSFT-filled wells, and left 20 minutes only in the last one.
?. [Mounting (D3)]
Mounting:
?. [Mounting (D3)]
Take a microscope slide, and add a drop of ~30uL of Fluoromount-G (mounting medium) to the centre of it
While we usually do not, a spacer can be used to separate the sample from the coverslip, and to preserve the exact morphology of the sample. The absece of a spacer is not particularly destructive for early Gastruloids, but some damage can be seen for 168h and later timepoints.The spacer can be either bought commercially, or made with two layers of double-sided sticky tape. The drop of mounting medium would then be added within the area defined by the spacer.
?. [Mounting (D3)]
Transfer Gastruloids to the drop (see technique in "Guidelines")
You can transfer any number of Gastruloids to the same drop. Depending on the size of their size, this might however increase the chance that they cluster together (e.g. 144h, 168h). If this happens, you can try to separate them and distribute them more evenly with an eyelash tool,  being careful not to damage them.When big Gastruloids are used, it is generally recomended to do multiple slides with fewer Gastruloids, rather than one with many all clumping together.
?. [Mounting (D3)]
Gently drop a coverslip on top of the samples
To avoid trapping air bubble, deposit a small drop of mounting medium at the centre of the coverslip prior to use.
?. [Mounting (D3)]
Using the edge of a Kimtech wipe, absorb out excess liquid seeping out of the coverslip edges
?. [Mounting (D3)]
Seal all sides of the coverslip with Permount hardening resin (we filled an empty nailpolish bottle with Permount, and use the brush for handling)
Nail polish can be used instead of Permount, but there are reports of alcohol possibly being able to seep into the mounting medium and reducing the long-term shelf-life of the sample.
?. [Mounting (D3)]
Keep the slide in a box away from light,  , until ready for imagingNOTE: slides can be kept at  for months before imaging with little apparent loss of signal. Try however to image slides as close as possible to when they were made
4 °C
4 °C