Fixation and staining of gemmule-hatched Ephydatia muelleri for fluorescence microscopy

Add 3-4mL volume of culture medium to each dish, and place 1-2 gemmules in the center of the inner well.

Grow the sponges for , in the dark (this reduces the growth of Chlroella-like algal symbionts that autofluoresce (particularly in the far-red channel).

Remove the culture medium from the outer well by pipetting or aspiration. Then, carefully remove the residual medium from the inner well using a p200 pipette to avoid damaging the tissue.

Gently add 2mL of fixative (4% formaldehyde in 95% alcohol) to the outer edge of the dish to avoid disrupting the sponge tissues.

Replace the lid to the dish and incubate at Room temperature for 0h 45m 0s

Remove the fixative by carefully pipetting from the outer edge of the dish only. (It is better to leave the fixative in the inner well undisturbed to avoid damaging the tissue).

Add 3mL of PTw to the outer edge of the dish, and incubate for 0h 3m 0s at Room temperature . Remove, and repeat.

Add 3mL of Block Solution to the outer edge of the dish, and incubate for 0h 45m 0s at Room temperature .

Dilute your primary antibody at an appropriate concentration in Block Solution. You will need 80-100µL of diluted antibody per sample.

Gently remove all Block Solution from the outer and inner well of your samples. It is important to remove all residual block solution so that you don't further dilute your primary antibody to an unknown extent.

Add 80-100µL of the diluted primary antibody solution to the inner well of the dish, being careful not to pipette directly onto the sponge tissue.

Incubate for 1h 0m 0s at Room temperature . Alternatively, you can place the sample at 4°C .

At the end of incubation, it is not necessary to remove the primary antibody by pipetting. Instead, simply add 3mL of PTw to the outer edge of the dish. Incubate 0h 5m 0s at Room temperature . Repeat 1x.

Dilute Hoechst and Phalloidin stock solutions to 1:100 [final concentration], and (if antibody staining) the secondary antibody conjugate to 1:500-1:1000 [final concentration] in Block Solution. You will need to prepare at least 80-100µL of this mixture for each sample. Protect this solution from light.

Carefully remove the final primary antibody wash from the outer and inner wells of the dish by pipetting.

Add 80-100µL of the Hoechst/Phalloidin/secondary mixture (Staining Solution) to the inner well of the dish.

Incubate in the dark, for 0h 45m 0s at Room temperature .

It is not necessary to remove the Staining Solution from the inner well. Instead, add 3mL of PTw to the outer well area, and incubate in the dark for 0h 3m 0s . Repeat 1x.

Remove the PTw wash from the outer and inner well area of the dish by carefully pipetting.

Add 80-100µL of mounting medium to the inner well of the dish.

Store the sample at 4°C in the dark until imaging.