Bouin's solution, may be. 150 ml concentrated water solution of picric acid + 50 ml formalin (40% solution) + 10 ml absolute acetic acid. Two acids (three in fact - formic acid in formalin too) must be decalsify the daphnia during 3 and more weeks. I used this trick with sea stars.

I feel that it will be a problem to cut Daphnia on microtome. Maybe you can use our methodology which was prepared for rotifers:

Article The Temperature–Size Rule in Lecane inermis (Rotifera) is ad...

Best regards, Ania

I think You can try used Technovit 7100. Now I work with plants, but try this technique on animal tissue and works great.

Thanks for all of your suggestions! Really appreciate it! I went on to fix my daphnia with Bouin's fluid and followed by decalcification using 6% formic acid. Will find out when the stained slides come back.

Two suggestions: If your results are satisfactory, of course, stay with your protocol. If not, I can suggest the following: We also worked with Daphnia, albeit not for paraffine embendding. We fixated them by either 4% formaldehyde in 70% EtOH, or by 1/2 Karnovsky´s fixative (2,5% Formaldehyde, 2% glutardialdehyde in buffer) with good results. For decalcification, you could also try EDTA, as it may be less harsh on tissues/cell nuclei. Once decalcified, You could also embedd them in plastic resin instead of paraffine, if paraffine proves difficult (infiltration/sectioning might be difficult due to the cuticle). e.g. LR White as an acrylic system, or AGAR LV as an epoxy system result in harder blocks, so you might get thinner sections (with glass/diamond knives), resulting in weaker staining, but enhanced resolution in micrographs.

Hi Paavo, thank you for sharing your protocol. I have a lot of daphnia, will definitely give it a try!

Hi Mee Siing Ngu,

I just remembered: Plastic embedded material stains a lot better, if you soften the plastic by placing the object slides in a chamber with ethanol fumes (EtOH-soaked paper on the bottom) at room temperature for 30 min. directly prior to staining. In case you used osmiumtetroxide (as for TEM-material), it helps to de-osmificate the slides in 10-15% H2O2 for 10-15min between softening and staining. That helped me to get much better stainings from epoxy-embedded material.

Good luck with your Daphnia, these are really nice objects

Cheers

Hi Paavo,

Slides of daphnia fixed in Bouin's solution just came back and they look fine (according to colleague who works on zebrafish histology). I am not really sure about that because we have nothing to compare to and there is so little of daphnia histology out there. Do you have any slides that you can share with me so I can use as reference? By the way, I am going to try few more fixatives, including your suggestions, first using paraffin embedding and eventually plastic embedding.

Thanks again for your time and suggestions! Really appreciate it!

MeeSiing

Dear MeeSiing,

we are a service facility for the Department of biology, and some of our customers were working with Daphnia, but unfortunately, I do not have histological slices of my own that I could share with you. I am sorry, I surely would have if I had some. Did you try the extensive work "Microscopic Anatomy of the Invertebrates", edited by Harrison & Ruppert? It contains a wealth of information on most groups of animals, has great images for comparison and is a valuable literature source, also for older high-quality references. Even if there is not too much about Daphnia itself, you will get some kind of idea what to expect.

http://www.worldcat.org/title/microscopic-anatomy-of-invertebrates-vol-9-crustacea/oclc/29358081

Then there is a german textbook on histology of Invertebrates. It does not contain much about Daphnia, and I am not aware of an english issue, but it contains many colour images of histological sections of different invertebrates, so you could compare what they generally look like ( there are some images of Astacus, for example)

http://www.worldcat.org/title/mikroskopische-anatomie-der-wirbellosen-ein-farbatlas/oclc/31306183

I wish you all the best, and good success with your Daphnia project!

Cheers

Paavo

Dear Paavo,

Thank you for taking the time to send me all the information. I really appreciate it! I am looking for books and I am sure these are going to be helpful. I have just found an article which is about fuctional morphology of Daphniidae. Maybe one of your customers would find it useful.

http://rstb.royalsocietypublishing.org/content/royptb/331/1259/1.full.pdf

All the best to you too!

Thanks,

MeeSiing

Dear MeeSiing,

Thank you very much for the link to the article. That is indeed very interesting, I will also send it to our costumers. Thank you for pointing me to it.

All the best to you, cheers

Paavo