As one can see from the answers, this is a complex issue that is generally guided through with some very basic instructions that have been already outlined. A factor I always take into account (and I have no idea whether that makes any sense, is the rule that with every 10C the reaction time doubles, i.e. if yo do the incubation overnight at 4C you can get the same result at room temperature in about 4 hours. But in reality you do not need most of the time an overnight incubation and your commercial antibody is "done" in less than an hour. But as stated by the others, it depends on the quality of your antibody and the expression level of the protein in your tissue, the preparation of the tissue etc.

I actually do by now all my incubation at room temperature over night (I use FFPE tissue, cooked for a while in a pressure cooker and sterile filtered solutions, not much of a danger for bacterial growth while incubating). There is no difference to incubating at 4C in regard to background with the antibodies I use. Background is mostly an indication of a bad antibody (or better antibody mix in case of polyclonal sera). 

So in the end, the incubation time, antibody concentration and incubation temperature are relatively irrelevant than kept in a certain range: the key is: is your antibody any good and you can find out with decent positive and negative controls and a good knowledge about the expected expression pattern (is the staining nuclear, cytoplasmatic, membraneous etc; restricted to certain cell types etc.) If you have no clue, it will be hard to evaluate the staining or the lack of it.

The best control for your staining is another antibody against the same protein, but another epitope. Both antibodies should deliver a similar pattern (in a near perfect world). There is no other way to tell if your staining is good or not. You can do isotype controls, throw on the peptide used to make the antibody, use phosphatase for phospho-antibodies etc etc. These negative controls are all fine (and should be done by the company that sells the antibody!), but they don't tell you whether the staining you see on your slides is really detecting the protein your are interested in. Even Western data are relatively meaningless since they detect are very very denatured protein while on your slides you may not have the same type of epitope.

I am not sure I am making a clear comment here. The point is, you should get decent results with your antibody on a relatively broad range of incubation times and temperatures. These conditions can be optimized. But you can't optimize a bad antibody, except making it worse and optimizing for background and unspeciific staining of unrelated epitopes. That is there your focus should be.

hi Arbind,

It's dependant on the antibodie you used. On my side usually i begin with an overnight incubation a 4°C for the primary antibodies and a 2h incubation for the secondary but sometime i have to adapt these incubation time. I you purshased a commercial one, you should have some documentation with it.

Johan

it depends on the titer of and the affinity of your antibody used. And it depends on the amount you have available and/or the costs of the antibody. In clinical histolab with IVD antibodies the incubation time for the primary is about 20-60 min and the secondary (commercial kit) is about 10 min. The titer is chosen so high to reach short incubation times, this is never an end-point-staining. Reaction with a low titer over night in the fridge may lead to a clear and end-point-staining, or may reveal also small amounts of antigen in the tissue. So the answer is (like often) it depends ...

Practically, I would suggest you use a commercially available detection system. This should give you instructions for optimal use. For the primary antibody, start with 30 min incubation at RT and do a dilution series. Again, the supplier should give you an indication as to what sort of dilution you are likely to be successful with. If you have no clue at all, cover a range (e.g., 1:50 to 1:500 for mouse monoclonal and even more dilute for polyclonals). If you find you have too much background staining, try the same overnight at 4°C.

This depends on many factors.  If the antibody you are using is commercially available and there are published protocols using this specific antibody in the same target species you should likely begin with what has been described and see how the results compare with what you can do in your lab.  Overnight refrigerated runs are often useful for low-affinity antibodies or expensive antibodies where you want to maximize your reagents. In the end, you want to use the dilution and time that yields the best staining for your application while minimizing background.  I typically create an optimization table that covers a range of dilutions and incubation times when starting out with a new antibody to determine the best conditions for our lab and reagents.

I prefer to apply it in 4 degree overnight. 2h is at risk.

As one can see from the answers, this is a complex issue that is generally guided through with some very basic instructions that have been already outlined. A factor I always take into account (and I have no idea whether that makes any sense, is the rule that with every 10C the reaction time doubles, i.e. if yo do the incubation overnight at 4C you can get the same result at room temperature in about 4 hours. But in reality you do not need most of the time an overnight incubation and your commercial antibody is "done" in less than an hour. But as stated by the others, it depends on the quality of your antibody and the expression level of the protein in your tissue, the preparation of the tissue etc.

I actually do by now all my incubation at room temperature over night (I use FFPE tissue, cooked for a while in a pressure cooker and sterile filtered solutions, not much of a danger for bacterial growth while incubating). There is no difference to incubating at 4C in regard to background with the antibodies I use. Background is mostly an indication of a bad antibody (or better antibody mix in case of polyclonal sera). 

So in the end, the incubation time, antibody concentration and incubation temperature are relatively irrelevant than kept in a certain range: the key is: is your antibody any good and you can find out with decent positive and negative controls and a good knowledge about the expected expression pattern (is the staining nuclear, cytoplasmatic, membraneous etc; restricted to certain cell types etc.) If you have no clue, it will be hard to evaluate the staining or the lack of it.

The best control for your staining is another antibody against the same protein, but another epitope. Both antibodies should deliver a similar pattern (in a near perfect world). There is no other way to tell if your staining is good or not. You can do isotype controls, throw on the peptide used to make the antibody, use phosphatase for phospho-antibodies etc etc. These negative controls are all fine (and should be done by the company that sells the antibody!), but they don't tell you whether the staining you see on your slides is really detecting the protein your are interested in. Even Western data are relatively meaningless since they detect are very very denatured protein while on your slides you may not have the same type of epitope.

I am not sure I am making a clear comment here. The point is, you should get decent results with your antibody on a relatively broad range of incubation times and temperatures. These conditions can be optimized. But you can't optimize a bad antibody, except making it worse and optimizing for background and unspeciific staining of unrelated epitopes. That is there your focus should be.

I think most is already said: It depends on the antibody and on the reaction temperature. 30 minutes to 2 hours at room temperature will already be fine for many (commercial) antibodies, others need over night at room temperature (home-made antibodies), with 4°C over night you will have good results for almost all antibodies.

You might also consider, what is more important: Time or money. If incubating at 4°C you might be able to use the antibody solution several times or use lower concentrations of the antibody. But of course it takes longer and if you need the results quickly...

Generally, incubation of the primary antibody for 1 hour and secondary antibody for 30 minutes both at 370C gives a good result especially when you use heat antigen retrieval method. The most sensitive part of this experiment is the dilution factor for both the primary and secondary antibodies and for this reason, you will need to have a checkerboard dilutions for your optimization. From this you can pick the dilutions that gives you the best result.

Good luck

It's not easy to respond to this question. It depends on many criteria:

- type of tissue

- nature of antigen

- nature of antibody (monoclonal, polyclonal) and its tiration as well as its specificity,...

To my opinion, you have to precise more your request 

You will need to work out conditions based on the type of tissue, how the tissue is prepared, specificity of the antibody and availability and location of the antigen.  If you have plenty of tissue I would try 2-3 protocols side by side and compare results.  The more information you have on your antibody the easier this will be.  I wouldn't go more than 1 hour with the secondary as it may increase background and most of the binding probably occurs in the first 10 minutes anyway.  Make sure your secondary solution has normal serum from the source of the secondary antibody.  For instance if your secondary is made in goat then add 5% normal goat serum to the secondary to block non-specific binding and increase your signal to noise.

It depends on type of tissue and how abundant of the proteins of interest.

You can do a pilot study; try different time points (1-3 hr) or overnight and different dilutions of primary antibody to optimise the condition.

The Immunohistochemistry staining is a little bit of trail and error method. It depend so many factors like type of tissue, Primary antibodies , secondary antibodies and detection system. So for this you have to standardized the protocol. . Generally primary antibodies will be either 1/100 or 1/50 for 1 to 1 and 1/2 hrs at 37 o C but  secondary antibodies depend upon factor like conjugated to FITC or Peroxidase. Please see the recommendation of dilution by the company and dilute 1 fold less i,e it it is written 1/5000 , you use 1/2500. You have make 4 sets of slides for standardization (two set for time and two set for dilution) . Finally you will get a combination which specific slide is giving better result and follow that.

Normally you can dilute more than what the company proposes, not less. If the company finds out that dilution between 1:500 and 1:1500 work for them, they will tell you to work with a 1:500 dilution - just because you need three times more antibody and they earn three times more money. (And maybe because the signal is stronger/incubation time is shorter).

Dear Dr Chaudhary as far our laboratory is concerned for primary antibody we incubate either for 1 hour at room temperature or at 4 degree Celsius overnight. In most of the international protocols incubation overnight is preferred and it gives good results, but at the same time is time consuming.Whereas, for the secondary antibody it depends upon the manufacturer, generally it is for 30 minutes at room temperature.If you need any other information you are most welcome.

In most cases, Pri. Ab- overnight at 4 degree and Secondary at RT for 1-2 hrs.

Hi,

If your primary antibody is at 1/100-1/400, you can incubate for 2h. More it's diluted, more the time of incubation is important (1/1000 : overnight). For secondary antibody, use a concentration of 1/100 or 1/200 (1-2h). Note that you have to rinse between the two incubations (3 x 10 min).

Thanks

Thomas Andl

You need to optimize conditions in your lab.  Always perform a optimization run on control tissue prior to starting your assay on test tissues.  As for time/temp 4oC O/N, 1-2hr RT, or 30min 37oC for primary Ab are routinely trialed by me, depending on concentration Ab/priceof Ab.  Secondary Ab depends on what you are using...  biotinylated, micropolymer system...fluorescently tagged.  You are always evaluating the signal to noise of the sections.  Optimal IHC run will give you very little background with intense reaction product localized to the correct cells/ cell compartment, i.e. nuclear, cytoplasmic, membrane bound.  refer to your Ab spec sheets for guidelines, and start there.

Although some protocols offer that primary antibodies incubation 4C overnight for immunohistochemistry, we forgot to put to samples at 4C and they stayed at RT(room temperature) overnight. Then, we improved a procedur that gives same results with standart protocol.

In case the samples with primary antibodies are left in RT overnight, you can try to get rid of unspefic bonds in order to continue to work with same samples. We faced with the condition, that accidentally left antibodies overnight in RT instead of 4 degrees. We designed and perfomed following procedur:

-2*15 minutes washing with %3 Triton 10x (or %0.3 Triton 100x) in TBS 1x [Because Triton provides getting rid of unspefic bonds as a detergent.]

-4*20 minutes washing with TBS 1x

After adding secondary antibodies we need to wait 1 hour in RT for incubation. We observed samples at Zeiss ApoTome and results were as clear as samples which we performed with standart protocol before.

Note: Our primary antibody was rb-antiRFP.

I just wanted to second Doğukan Koçyiğit 's answer - we have applied our anti-c-fos antibodies in 50um brain sections overnight at RT. I based that decision in our lab due to reading another protocol applying a pSTAT3 antibody at RT for such an extended time period. At least for c-fos, this RT lengthy incubation definitely improved the staining signal. Also in our case, we don't do any "pre-block"/permeabilization steps beforehand, but we do have 2.5% normal serum, 0.3% triton, and 0.05% azide in PBS as the primary antibody diluent - the azide may be minorly important for preventing microbe growth at RT. I get the impression that what leads to more non-specific binding is the (lacking) quality and specificity of an antibody more than incubation time, or perhaps something about the tissue that prevents binding to the epitope of interest. In any case, after that primary we still do a secondary with HRP and use tyramide signal amplification to 1. mimic old-style DAB staining of c-fos and 2. make it visible at 5x, where everything in a section that thick is still in focus and quicker to capture sequential images on the microscope.

A side note - never combine azide with HRP in the same solution, otherwise HRP becomes non-functional.

We use 30 min for Primary antibody at RT and Dako secondard antibody conjugating system. You should try any of above given incubations according to your needs.

For my experiment with leptin receptor antibody:

primary antibody = incubate at 4 degree Cen. over night.

Secondary antibody = 1 hour at room temperature.

However, read the peculiar protocol of your antibody to be more specific...

Well, in general. it should be not more than 1 hr each for primary and secondary. More important than the time is the temperature. You should always do the incubation in a normal incubator set at 42 degree Celsius. This temp. is essential for the proper and highly accurate binding of the antibody to the antigen. The antibodies of the immune system works best at 42 degree Celsius, so your antigen-antibody binding will be extremely efficient at 42 degree Celsius. I never liked the incubation at 4 degree Celsius overnight putting the whole IHC inside a freezer. I think this 4 degree Celsius gives rise to a lot of non-specific binding. If the antibody does not show any staining at 42 degree Celsius for 1 hr, then most of the time the antibody is no good for immunocytochemistry and suggests that the epitope is hidden and inaccessible in vivo or covered by other proteins. This has been my observation for the past 20 years! Try to find a good primary antibody that works in paraffin section immunocytochemistry incubating at 42 degree Celsius for 1 hr and then you will see all your problems will disappear.

Regards

Personally, I never heard about incubation temperatures above 37°C. Typical temperatures are 4°C, room temperature and 37°C.

In general antibody-antigen reaction is an on-off-system. The better the match of antigen and antibody the longer it stays on the "on"-status. At higher temperatures the molecules move faster, giving antibodies a higher chance to meet an antigen. In the cold you need much more time but therefore the reaction might be more specific as antibodies have time to find the optimal partners and don't rush to bind to the next best protein.

For most primary antibodies 0,5-1 hour at room temperature combined with thorough washing to remove non-specific binding should be fine. Commercial secondary antibodies are well optimized and have an easier access to their targets so the time for secondary incubation may be even shorter.