Technique / Histology / Immunohistochemistry techniques
Basic Immunohistochemistry Protocol
Basic Immunohistochemistry Protocol
Placed on WWW by: Michael Serfas
General Note:
Protocols for immunohistochemistry vary widely, due to the differences between antigens and their recognition by antibody. Some epitopes are destroyed by the high temperatures and organic solvents used in paraffin fixation, while others cannot survive freeze and thaw. Each fixative works by a different mechanism, which may either help to uncover the epitope, or may destroy it or make it less accessible. Some antibodies may even adhere to the blocking agent used, so that it causes rather than ... ...
URL: Basic Immunohistochemistry Protocol
Contents: original article
Basic Immunohistochemistry Protocol
Placed on WWW by: Michael Serfas
General Note:
Protocols for immunohistochemistry vary widely, due to the differences between antigens and their recognition by antibody. Some epitopes are destroyed by the high temperatures and organic solvents used in paraffin fixation, while others cannot survive freeze and thaw. Each fixative works by a different mechanism, which may either help to uncover the epitope, or may destroy it or make it less accessible. Some antibodies may even adhere to the blocking agent used, so that it causes rather than eliminates background. For success it is crucial to try a number of different techniques and compare the results. If at all possible, obtain all details of the procedure used by other researchers for that antibody with successful results; this can save much effort.
Once a "signal" is obtained, proving that signal truly reflects the distribution of the molecule of interest is still a matter of some difficulty. The simplest negative control is the absence of expression in tissues in which the RNA for the molecule of interest is known not to be expressed by RNase protection, and restriction of expression to regions of a target tissue that have expressed RNA as seen by in situ hybridization. A better negative control is the elimination of the signal by pre-incubating the antibody with an excess of the peptide or protein with which it was raised. A Western blot can suggest specificity of the interaction if only one band is seen; nonetheless, the difficulty of optimizing conditions for a Western blot should demonstrate the possibility that conditions in the immunohistochemistry reaction are less than optimal. On the other hand, there can also be doubts as to whether the antigen is visualized everywhere; in particular, one must determine whether the fixation protocol used actually permeabilizes the nuclear membrane to a sufficient degree for nuclear antigens to be fully visible.
This protocol represents the basic procedure recommended for proteins present in relatively high abundance. We have also made use of a more sensitive protocol, using Triton X-100 permeabilization, and gold chloride/silver developer for signal intensification.
Paraffin sections:
After dissecting tissues, fix in 2% paraformaldehyde, Bouins solution, or other fixative from 30 minutes to overnight. Larger tissue sections require longer periods of time; the time of fixation will affect the characteristics of sectioning the tissue (crumbling or cracking of the tissue) and possibly antigen recognition. In any case, the tissue will be exposed to ethanol for a prolonged period during dehydration, which itself causes some fixation. A basic paraffin embedding protocol is given in the Lee lab protocol for in situ hybridization. Section the tissues 5-10 micrometers thick.
Incubate 2-3 times in xylene for 10 minutes each.
Incubate twice in 100% Ethanol for 2 minutes each.
Hydrate by placing in 95%, 70%, 50%, 30% ethanol for 2 minutes each.
Optional: If endogenous peroxidases are a problem in this tissue type, and you are visualizing antigen with horseradish peroxidase, then inactivate the peroxidase by incubation for 5-15 minutes (longer for paraffin tissues) in 0.1% hydrogen peroxide in PBS (Santa Cruz protocol) , or 30 minutes in 0.3% hydrogen peroxide in methanol (Vectastain kit). Afterwards, wash in buffer (3 changes) or running water for 20 minutes. For my studies in intestinal sections, this did not appear to be necessary.
Place slides in buffer for 5 minutes. Buffer: 0.25 M Tris-HCl at pH 7.5; other protocols often use PBS.
Optional: Post-fix slides for 2 minutes in the original fixative used for the paraffin slides. This decreases the tendency of the sections to detach from the slide --- slightly. In general, treat paraffin sections in this procedure GINGERLY. Even if they do not completely detach, the tendency of edges of the tissue to lift up allows antibody to become trapped beneath the edges of the tissue, causing high background.
Optional: Block slides with 10% serum from the species from which the secondary antibody was taken. Incubate for 20 minutes at room temperature in a humidified chamber. Wash in buffer for 5 minutes.
Place slides into buffer containing 0.5% BSA and 2% Fetal Calf Serum for five minutes [note: the biotin present in this small quantity of FCS did not appear to interfere with recognition of biotinylated antibody].
Incubate slides in a humidified chamber overnight with primary antibody. To create the "chamber", use cut disposable pipettes to form a support for the slides above H2O saturated paper towels in a large petri dish; cover for incubations). Dilute antibody in the Tris with protein solution above. Always dilute the antibodies in a solution containing protein! Many other protocols use 3% BSA or the like. Antibody is usually diluted from 1:20 to 1:200; as with so many parts of this procedure, this must be determined empirically for a new antibody. Use Kimwipes to remove excess buffer from the slides, then add 50-60 microliters of antibody to the slide. The antibody should be restricted to the wet region of the slide by surface tension alone; if you have trouble, "PAP Pens" and other hydrophobic markers are sold by many companies.
Rinse slides first with a gentle stream of buffer from a squirt bottle, that flows across the sections from above.
Wash 5 minutes in buffer.
Block 5 minutes in Tris with protein.
Blot slides as before, and incubate with secondary antibody in a humidified chamber for 30 minutes or longer.
Rinse from squirt bottle and wash 5 minutes in buffer as before.
Fluorescence visualization:
The secondary antibody will usually be labeled with FITC or TRITC, so the slides can be mounted after this rinse. Mounting should be done in an aqueous medium. Gel/Mount, available from Fisher, preserved my slides for over a year before they began to noticeably degrade; however, the sharpness of the image was not quite as high as with DAB and Permount. Temporary glycerol mounts, in my hand s, produced much light scattering and very poor image quality.
Peroxidase visualization:
Mix 2 drops Vectastain "A" and 2 drops Vectastain "B" in 10 ml PBS as per kit instructions at the beginning of the secondary antibody incubation (30-60 minutes before use).
Incubate 1 hour with peroxidase-anti-peroxidase mix in a humidified chamber. Rinse with buffer as before.
Incubate in fresh DAB solution. (10 mg DAB + 20 ul 38% H2O2 in 20 ml 0.1 M Tris pH 7.2; filter; optionally, add 200 ul 1 M imidazole) Stop the reaction by washing in running water when a uniform brown color first becomes visible on the section.
Frozen sections:
Freeze and section as in the Tyner lab in situ protocol. Fix in one (or more) of the following ways:
5 minutes in Bouins at room temperature.
10 minutes in acetone at 4 degrees C.
15 minutes in methanol at -20 degrees C.
2 minutes in 2% paraformaldehyde at 4 degrees C.
2 minutes in 4% paraformaldehyde at 4 degrees C, then 10 minutes in 70% EtOH at 4 degrees C, then dehydrate (as per in situ hybridization; a starting point for combined protocols?).
Incubate in three changes of fresh buffer (2 minutes each).
Proceed with primary antibody as for paraffin-embedded sections.
Last update 11-Mar-2002, Rating of 21 votes.
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By viji
on 09-Feb-2009
you check with your antigen retrieval step. If you are using microwave method, u better try with pressure cooker and also check whether the buffer you use (mostly citrate ph 6 is used for many markers) is appropriate one for the your protein of interest |
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By Dr Dilip Kumar Swain
on 30-Jun-2008
Dear sir, I am a research scholar in the neurophysiology lab in AIIMS, India. Currently I am working in the field of spinal cord injury and gene expressions. I want to study the differential gene expressions in the damaged spinal cord. Sir kindly provide me the protocol for this aforesaid |
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By indrajeet
on 27-Oct-2007
I have problem that my section started cracking after its visiolised by DAB protocol. I do not understand why its cracking as it was immerstion fixed tissue. |
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By Matthew
on 23-Oct-2007
The site''s very professional! Keep up the good work! Oh yes, one extra comment - maybe you could add more pictures too! So, good luck to your team! |
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By skariah
on 23-Sep-2007
Dear sir, |
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By Ranga wittachchi
on 19-Sep-2007
I'm doing a IHC to detect k-19 antigens on breast cancer tissues.I use human k19 specific mouse monoclonal antibody as the primary antibody and R&D kit for the following steps.I have done 1:50,1:100,1:200 qnd 1:500 primary antibody dilutions.Unfortunately 1:50 and 1:100 sections give dark background with the staining.the intensified brown colour is clearly visible in the naked eye but stained and unstained cells can not be dicriminated the by the microscopic view.1:200 and 1:500 do not give this coloured background but the do not stain K-19 specific cell in the positive control samples as well. |
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By Qutaiba Alrawi
on 08-May-2007
Hi Dr |
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By Qutaiba Alrawi
on 08-May-2007
Dear Sir |
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By Romina
on 10-Jan-2007
I am doing immunohistochemistry in rat embryos but I cant obtain any mark. I try dilution from 1:20 to 1:200 and incubation time from 1h to 24 over night. Should I try a 48 hs incubation +4 ºC or doesn`t have any sense? |
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By D.R.Karthikeyan
on 04-Dec-2006
dear sir, |
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By yaping zeng
on 13-Nov-2006
Do you have the protocol for immunostainning for culture cells with Cx43, Cx37,Cx40. |
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By Jennifer C
on 30-Jun-2006
Dear sir, |
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By Shahab
on 03-Jun-2006
i m having problem in getting paraffinsectioning of substantia nigra pars compacta send me some protocol to get these section with the help rotatory microtome |
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By dr P.shahsikala
on 31-Jan-2006
hi |
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By Andreea
on 05-Jan-2006
Hi! |
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By Debraj Roy
on 30-Oct-2005
Sir |
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By Bunyanuch
on 21-Sep-2005
I try to detect germ cell in ovary of crocodiles so I want to know primary antibody and secondary antibody if I use alkaline phosphatase. I use indirect immunohistochemistry by fixed in Bouin's solution. |
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By shweta
on 05-May-2005
hi, |
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By Valerie
on 23-Feb-2005
Hi, Thanks very much for all the info. I would like to know if fluorescence detection for paraffin slides is as good as HRP detection |
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By Jeff Lakritz
on 31-Jan-2005
I am having trouble with either endogenous peroxidases or biotinylated proteins in lungs because I get similar background in both control and Positive tissues. Should I just try alkaline phosphatase labeled secondary or treat slides with polylysine or raise pH of ABC reagent? |
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By Sandie Jacobi
on 06-Jan-2005
I am trying to do IHC on fix-inflated lung (paraformaldehyde), soaking in 30% sucrose, then freezing in OTC media and then doing cryostat sectioning. I am using an HA-tagged anti-body and seeing possitive stained airways in my negative controls. I would like to talk with you about some options. Please respond to my email when you can. Thank you, Sandie |
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By Tatiana Gartner
on 02-Jul-2004
dear, |
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By zita
on 28-Jan-2004
Dear Sir, |
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By jelena
on 18-Nov-2003
Dear Sir, |
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By zaza
on 22-Nov-2002
Dear Mr. Serfas, |
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By Nathan Elam
on 07-Aug-2002
Dear sir, |
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By senthil kumar.c.
on 13-Jul-2002
sir i am performing IHC for ras p21 in paraffin sections iam not getting proper results.can u suggest me the best protocol for ras p21 IHC |
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By bella
on 21-May-2002
Dear Sir, |
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By Gulper?Oktem
on 17-Apr-2002
do you have any protocol for stainig culture cells. Thank you. |
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By archna
on 11-Mar-2002
Dear sir |
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