Hi Jeffrey,

what species are you testing this antibody on? Are you using free-floating sections? How thick? What secondary antibody have you used to visualise 5-HT?

I'm staining in Long-Evans hooded rats, roughly 12 Weeks old. Sections were made last week, very fresh. Sections were sucrose cryoprotected and sectironed in a cryostat. They're currently mounted to slides with no mounting medium. They were sliced at 10 microns. I used a goat anti-rabbit (546 I'm pretty sure, I'm out of the lab right now and can't check) secondary 

I think there is something wrong in your reaction, and I am not sure I would trust weak labelling at 1:1000, given that 1:25000 seems to be a working concentration for many people. How long did you incubate for (both secondary and primary), what concentration of secondary did you use?

I incubated my primary overnight (18-20 Hours) and my secondary 2 hours (although this time it'll be one hour and then amplification) at a 1:400 dilution.  

You could try reincubating with the primary at 4°C for 48 hours (or over weekend), as indicated in the description of this antibody. Then incubate for 2 h at RT with the secondary,

Why are you using amplification? Staining seems to be bright enough for other users, as long as the fluorophore is bright (594, Cy3, 488).

For example, have a look at the following protocol published in the reviews of this antibody:

Tissue preparation for rat tissue:

Transcardial perfusion using 4% paraformaldehyde, post fixed overnight in 4% para, and cryoprotected in 30% sucrose for 3 days. Freeze tissue, sectioned (at 40 micron), and use immediately within days.

Indirect Immunofluorescence staining protocol:

Day 1

1. wash 1x PBS for 15 min

2. blocking solution (1x PBS + 0.3% Triton X100, 3% goat serum) for 1 hr at RT

3. Incubate tissue sections to primary antiserum (rabbit anti-5HT, ImmunoStar #20080) diluted in blocking buffer overnight @ 4';C.

Day 2

5. wash tissue sections 3 x 10 min with 1xPBS.

6. Incubate tissue sections with secondary antibody (e.g. goat anti-rabbit-Alexa Fl 488, Jackson ImmunoResearch Laboratories, Inc., diluted 1:500 in PBS) for 1-2 hrs in the dark at room temperature.

7. wash tissue sections 3 x 15 min with 1x PBS in the dark.

8. Coverslip with vectashield and store at RT.

I know your time is limited, but I would consider another titration with concentrations both lower and HIGHER than 1:25000.

The protocol looks pretty similar to the one I'm using. I'll look into different levels if time and resources will allow for it. I'm using the amplification because my fluorescence I got was very weak. My guess is that it's because my antibody is older, it's possible it has lost some level of effectivity during this time, but I'll double check details of reactions as well. 

Dear Jeffrey,

I read quickly through the data sheets and reviews for the antibody at the link you provided.  It looks like most people who use only a fluorescent secondary antibody for detection dilute the primary at 1:1000 to 1:5000.  The really high dilutions (1:20,000 or so) are recommended by the company for ABC/DAB.  That procedure amplifies the signal A LOT because of the number of enzyme molecules that can be coupled to the ABC complex (http://www.ncbi.nlm.nih.gov/pubmed/20012837).   In my experience, it is not unusual for there to be this magnitude of difference in dilutions when using a fluorescently labeled secondary antibody for detection vs. ABC/DAB. In addition to the savings in amount of primary antibody needed for a study, the ABC/DAB method is very useful since it is permanent and can even be detected at the EM level if needed.   However, if you don't have experience with the Avidin/Biotin system (since you can also get more background, you will need to buy a biotinylated secondary antibody and the reagents or kit to create the ABC complex, you have to worry about endogenous biotin in tissues, DAB is a carcinogen, etc), I would stick to lower dilutions of the primary and a really bright fluorescent anti-rabbit secondary.

Do you have/ are you able to borrow another rabbit polyclonal antibody that will recognize some epitope in your tissue that you can use as a positive control for the secondary antibody?  ( For example, the Dakopatts rabbit anti-GFAP will label astrocytes really well.)  I suggest that you take the time to make sure that the secondary is working well before titering the anti-5HT.  And I wouldn't be afraid just yet of too much background from a 1:1000 dilution, as long as you perform the necessary controls.

In my opinion/experience, antibodies are much cheaper than your time or animals in the study.  If your secondary is working well, I suggest that you buy a new lot of primary antibody, make sure that you take the time to reconstitute it for frozen storage, and freeze the aliquots right away (testing it on some tissue at the same time, of course).  That way, there will be no question whether it is too old to work well.

Good Luck!

Jill 

Hi Jill,

Thanks for your answer! I've since done the procedure since I only had a 2-day window to do it in using a 1:1500 Dilution and titration, which produced usable results. In the future I'll be sure to do that though!

Thanks to all! :)

The antibody in my experience worked at very dilute concentrations, but just picking one to try is not helpful You should a) use paraformaldehye-fixed material (if not already doing so) and conduct a full titration:  1:1000, 1:3000, 1:10,000, 1:30,000, 1:100,000 and 1:300,000. incubations should be for 48 h and staining with NiDAB or DAB for 20 minutes.  See this paper

Dear Jeffrey, Gloria made a very important point -  for best results you should always perform a full titration of your primary antibody. There are so many factors influencing reactivity of your tissue that information from the literature should be treated as 'guidance only'. The secondary antibodies, their tags and chomogens (in case of enzymatic conjugates) need testing, but in good labs you can usually rely on standard protocols available to all new members of the team. Good luck with your experiments.

Thank you to all, you've been very helpful :) 

Hi Jeffrey,

Have you solved your 5HT staining issue? I'm having similar problem with the 5HT antibody and wondering if you solved your problem

Hi Ganchimeg,

I did solve the problem, which was lucky because I had to go straight into it without testing because of time constraints. Ultimately what I did was used a dilution of 1:1500 and did titration. Depending on who you get your antibodies from of course your ideal concentration may vary a bit. This gave me very good staining with (remarkably) very little background. I would recommend doing a test with the titration at a couple different concentrations, but ultimately for me it was definitely useful and necessary to amplify the signal enough. Hopefully that will work for you!