TITLE: Fixation and staining of gemmule-hatched Ephydatia muelleri  for fluorescence microscopy
AUTHORS: Scott Nichols

[DESCRIPTION]
This protocol is intended for the preparation of gemmule-hatched freshwater sponges for imaging with an inverted scanning confocal microscope.

[STEPS]
SECTION: Fixation and washes
3. 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.
SECTION: Permeabilization and Blocking
8. Add 3 mL of Block Solution to the outer edge of the dish, and incubate for 45 min at Room temperature .
SECTION: Incubation with primary antibodies (if immunostaining)
9. 
 
Dilute your primary antibody at an appropriate concentration in Block Solution. You will need 80-100 µL of diluted antibody per sample.

 

SECTION: Counterstaining for DNA, F-actin
14. 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 80-100 µL of this mixture for each sample. Protect this solution from light.


 



SECTION: Mounting, storage, imaging
19. Remove the PTw wash from the outer and inner well area of the dish by carefully pipetting. 
SECTION: Plate gemmules in coverslip-bottom culture dishes
1. 
 
Add 3-4 mL volume of culture medium to each dish, and place 1-2 gemmules in the center of the inner well. 

SECTION: Plate gemmules in coverslip-bottom culture dishes
2. Grow the sponges for  , in the dark (this reduces the growth of Chlroella-like algal symbionts that autofluoresce (particularly in the far-red channel). 

 


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


SECTION: Fixation and washes
5. Replace the lid to the dish and incubate at Room temperature for 45 min

SECTION: Fixation and washes
6. 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). 
SECTION: Fixation and washes
7. Add 3 mL of PTw to the outer edge of the dish, and incubate for 3 min at Room temperature . Remove, and repeat.

SECTION: Incubation with primary antibodies (if immunostaining)
10. 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.
SECTION: Incubation with primary antibodies (if immunostaining)
11. Add 80-100 µL of the diluted primary antibody solution to the inner well of the dish, being careful not to pipette directly onto the sponge tissue.

SECTION: Incubation with primary antibodies (if immunostaining)
12. Incubate for 60 min at Room temperature . Alternatively, you can place the sample at 4 °C   .

SECTION: Incubation with primary antibodies (if immunostaining)
13. At the end of incubation, it is not necessary to remove the primary antibody by pipetting. Instead, simply add 3 mL of PTw to the outer edge of the dish. Incubate 5 min at Room temperature . Repeat 1x.

SECTION: Counterstaining for DNA, F-actin
15. Carefully remove the final primary antibody wash from the outer and inner wells of the dish by pipetting. 
SECTION: Counterstaining for DNA, F-actin
16. Add 80-100 µL of the Hoechst/Phalloidin/secondary mixture (Staining Solution) to the inner well of the dish. 

SECTION: Counterstaining for DNA, F-actin
17. Incubate in the dark, for 45 min at Room temperature .


SECTION: Counterstaining for DNA, F-actin
18. It is not necessary to remove the Staining Solution from the inner well. Instead, add 3 mL of PTw to the outer well area, and incubate in the dark for 3 min . Repeat 1x.

SECTION: Mounting, storage, imaging
20. Add 80-100 µL of mounting medium to the inner well of the dish.

 


SECTION: Mounting, storage, imaging
21. Store the sample at 4 °C in the dark until imaging.