perhaps this could help you

Methods Mol Biol. 2012;887:179-91. doi: 10.1007/978-1-61779-860-3_16.

Whole mount immunohistochemistry and in situ hybridization of larval and adult zebrafish dental tissues.

Verstraeten B1, Sanders E, Huysseune A.

Here is one paper for in situ hybridization on cryosections of adult retinas. 

http://www.ncbi.nlm.nih.gov/pubmed/10800703

Hi Kunal,

I have done Fluorescent RNA in situs on paraffin sections for adult zebrafish heads. Below is the link for the blog I wrote about it. 

https://insitutech.wordpress.com/2015/12/17/in-situ-hybridization/

Maybe this could help. Let me know, if you have questions. 

Best Simone

Thank you Mario, Nergis and Simone! Simone, your protocol is exactly what I was looking for as I also need to focus on the head region! Thanks a million! :) One other question that has now entered my mind is whether you sacrifice the adult fish via a specific method prior to fixing them. Please can you advise further?

Hi.

I found this on line. I guess you could use the ice before to fix the Danio rerio. Some "bad" people were putting directly the poor fish into the fix, please do not do it :-(.

/good luck

..................................................................................................................

INSTITUTIONAL ANIMAL CARE AND USE COMMITTEE (IACUC)

GUIDELINES FOR EUTHANASIA OF ZEBRAFISH (UPDATED June 2015)

The methods used must be specified in your approved IACUC protocol.  Acceptable methods of euthanasia are described in the AVMA Euthanasia Guidelines under the Laboratory Finfish section. 

Approved methods for adult zebrafish 7 days post fertilization (dpf) and older include:

Tricaine (MS-222): Immerse fish in a solution of tricaine methanesulfonate (Finquel or Tricaine-S). The solution should be buffered with sodium bicarbonate to a pH of 7.0-7.5. Stock preparation is 4g/L buffered to pH7 in sodium bicarbonate (at 2:1 bicarb to MS-222).  Euthanasia dosage 300ug/ml or 7.5ml stock solution to a total of 100ml. Fish must remain in the solution for 10 minutes following cessation of opercular (gill) movement.

Rapid chilling: Submerge fish in 0-4⁰ C chilled water (5 parts ice to 1 part water )   Fish should not be in direct contact with ice. Fish must remain in the chilled water for 10 minutes following cessation of opercular movement. 

Zebrafish fry 4-7 days post fertilization:

Tricaine or rapid chilling may be used as above, but fry should remain submerged in solution for 20 minutes following cessation of opercular movement

References:

American Veterinary Medical Association.  2013.  AVMA guidelines on euthanasia, 2013 update.

Koerber AS and Kalishman J.  2009.  Preparing for a Semiannual IACUC Inspection of a Satellite Zebrafish (Danio rerio) Facility.   Journal of the American Association for Laboratory Animal Science.  48: 65-75.

NIH. 2009. Guidelines for Use of Zebrafish in the NIH Intramural Research Program.

Wilson JK, Bunte RM and Carty AJ.  2009.  Evaluation of Rapid Cooling and Tricaine Methanesulfonate (MS222) as Methods of Euthanasia in Zebrafish (Danio rerio).  Journal of the American Association for Laboratory Animal Science.  48: 785-789.

NIH guidelines http://oacu.od.nih.gov/ARAC/documents/Zebrafish.pdf

Matthews M, Varga ZM. 2012. Anesthesia and euthanasia in zebrafish. ILAR J 53:192–204

Hi Kunal,

we are actually using a combination of the methods which Mario mentioned.

We use an overdose (see concentration from answers above) of Tricane to anesthetize/euthanize the fish. Once the fish stop moving, we keep the container with the Tricane and the fish in it on ice for 15 to 20 minutes. 

The last thing you want is the fish to "wake up" when they are in the fixative. Not only is it cruel, but it also can mess with the gene expression (up regulation of heat shock proteins due to stress response).

Cheers

Simone

Many thanks Mario and Simone! Your answers have finally ended my search of almost a month :) Also, as part of working with animals, I am required to take a Home Office License examination where we learn how to minimise suffering to animals. Glad to hear that researchers like yourselves put so much emphasis on lab animal welfare. Many thanks once again.