Dear Saurav:

I think I might give you some general guidelines even though I have not a specific protocol for catfish pineal organ wholemount immunofluorescence, and it has been a while since I worked on fish pineal organs.

First, I found that antibodies against serotonin and S-antigen were the most reliable pinealocyte markers. They labeled the vast majority of pineal cells, all of which where clearly photoreceptor cells when checked at the ultrastructural level. And they labeled the entire cells. Any well-documented commercial antibody against serotonin should do well; as for S-antigen antibodies, you will have to consult recent literature (it’s more than 25 years since I did this!).

Second, it should certainly be possible to perform whole-mount immunofluorescence on catfish pineal organs. For this it is an advantage that they are so small, and if they are difficult to handle because of the small size you could try embedding them in low-temperature gel-point agarose before the immunoprotocol.

I attach a suggested protocol outline.

Best of luck – it’s good knowing someone is still interested in fish pineal organs!

Peter

I don´t know much about pineal organ, but… have not you tried with immunohistochemistry or in situ hybridization in whole mount embryos?

Then, you can do gross slices of the pineal organ with a vibratome.

Ismael.

Dear Peter Sir,

Thanks for your valuable suggestion. My research field is fish pineal organ.

I need the ab against either serotonin or S-antigen. Can I use anyone or both (serotinin or S-antigen)? Thanks again for encouraging me.

With regards,

You can make a double-immunostaining to get a better charaterization.

Ismael.

Dear Ismael,

Kindly send me the double-immunostaining protocol. I am not working on fish embryo.

Thanks.

Hi again Saurav,

Didn't you get the double immunostaining protocol I sent you?

Peter

Hello Peter Sir,

Kindly sent me the double immunostaining protocol.

With Kind regards,

Saurav

OK I'll put it in the plain text too this time:

"Simultaneous visualization” of two antigens with indirect immunofluorescence

Example.: Mouse anti-S-antigen (monoclonal) and rabbit anti-serotonin, in small wholemount preparations.

NOTES:

If embedding the pineal organ in low-temp agarose, do it during or after step 2. Remove the agarose during or after step 13.

Rinsing and permeabilization times (steps 2-3) should be minimized – try shorter times and perhaps lower Triton X-100 concentrations once the protocol works!

Antibody concentrations (in steps 7 and 11) have to be tested and optimized!

Step 14: if you consider processing many pineal organs at the same time, and intend to use an inverted microscope-based confocal, you could mount the pineal organs in multi-well slides with coverglass bottoms.

I have never worked with whole mount embryos. I hope you could copy or download the protocol which have been sent by Sir Peter