Immunohistochemistry: Paraffin Sections Using the Vectastain ABC Kit from Vector Labs

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Chi, V., Chandy, K. G. Immunohistochemistry: Paraffin Sections Using the Vectastain ABC Kit from Vector Labs. J. Vis. Exp. (8), e308, doi:10.3791/308 (2007).

Abstract

Immunohistochemistry (IHC) is a valuable technique utilized to localize/visualize protein expression in a mounted tissue section using specific antibodies. There are two methods: the direct and indirect method. In this experiment, we will only describe the use of indirect IHC staining. Indirect IHC staining utilizes highly specific primary and biotin-conjugated secondary antibodies. Primary antibodies are utilized to discretely identify proteins of interest by binding to a specific epitope, while secondary antibodies subtract for non-specific background staining and amplify signal by forming complexes to the primary antibody. Slides can either be generated from frozen sections, or paraffin embedded sections mounted on glass slides. In this protocol, we discuss the preparation of paraffin-embedded sections by dewaxing, hydration using an alcohol gradient, heat induced antigen retrieval, and blocking of endogenous peroxidase activity and non-specific binding sites. Some sections are then stained with antibodies specific for T cell marker CD8 and while others are stained for tyrosine hydroxylase. The slides are subsequently treated with appropriate secondary antibodies conjugated to biotin, then developed utilizing avidin-conjugated horseradish peroxidase (HRP) with Diaminiobenzidine (DAB) as substrate. Following development, the slides are counterstained for contrast, and mounted under coverslips with permount. After adequate drying, these slides are then ready for imaging.

Protocol

Staining of paraffin sections

  1. Dewax slides with Xylene (Fisher X3S-4) 3x for 5 minutes each (change xylene every month depending on use, xylene is TOXIC). Use dishes and covers for 20 slides from Fisher ref 08-812-1A and racks made to fit these dishes. 200 ml liquid per dish.
  2. Hydrate through alcohol gradient as follows (change gradient every 2 weeks):
    • 2x in 100% ethanol (Fisher # A406-20) for 2 min each
    • 2x in 95% ethanol for 2 min each
    • 1x in 70% ethanol for 2 min
    • 1x in 50% ethanol for 2 min
    • 1x in 30% ethanol for 2 min
    • 1x ddH2O for 2 min
  3. Soak in DPBS for 5 min (DPBS = Dulbecco's modified Phosphate Buffered Saline with Ca2+ and Mg2+).

Antigen recovery:

  1. Pre-heat the Na-Citrate buffer (10 mM, pH 6.5) in the microwave.
  2. Cook the slides for 15 min in the microwave. The slides should never dry so check every min and add Na-Citrate when necessary.
  3. Let cool at room temperature.
  4. Block endogenous peroxidase activity with 1% H2O2 in DPBS for 20 min (1.5 ml of 30% stock Sigma H-1009 in 50 ml DPBS).
  5. Soak in DPBS 3 x 5 min.
  6. Block non-specific sites by incubating overnight at +4°C in DPBS + 5% bovine serum albumin (BSA) + 0.1% Na Azide + 5% serum.

    Note: Choice of serum will depend on species in which antibodies used for staining were made. Block with serum from the species in which the secondary Ab (conjugated to biotin) was made. If this is not available, use serum from a species different from the one in which the primary Ab was made.

  7. Block the biotin sites with the avidin/biotin blocking kit (Vector laboratories catalog # SP-2001):
    • Incubate with Avidin D for 15 min. Rinse briefly with DPBS.
    • Incubate with the biotin solution for 15 min.
  8. Incubate in primary Ab for 2 hrs at room temperature, in DPBS + 2% BSA + 0.1 % NaAzide + 2% serum (same as for Blocking step).
  9. Soak in PBS 3 x 5 min.
  10. Incubate with the secondary Ab conjugated with biotin for 1 hour at room temperature in DPBS + 2% BSA + 0.1% Na Azide + 2% serum (same as used for the blocking step).
  11. Prepare ABC reagent at the same time because it has to develop for at least 30 min before use!!! (see Materials)
  12. Prepare in a 50 ml conical tube. In 15 ml DPBS (no serum, no azide) add 3 drops of reagent A, mix and add 3 drops of reagent B and mix. Avoid squeezing the bottles, rather wait for the drops to form on their own. ABC kit Vectastain PK-6100 from Vector Labs.
  13. Soak in DPBS 3 x 5 min.
  14. Incubate with ABC reagent for 30-45 min at room temperature.
  15. Soak in DPBS 3 x 5 min.
  16. Prepare DAB peroxidase substrate (Vector Labs # SK-4100) in 5 ml ddH2O in a glass vial immediately before use (see Materials)
    • 2 drops of buffer stock solution, mix
    • 4 drops of DAB, mix (should become slightly brown)
    • 2 drops H2O2, mix
  17. Drop the DAB substrate on top of the slides and watch for brown staining.
  18. Dip slides into ice + tap water to stop the reaction.
  19. Rinse under cold tap water for 5 min.
  20. Counterstain with Hematoxylin (20 sec; Fisher CS401-D) and rinse in tap water until water comes out clear.
  21. Dehydrate through alcohol gradient starting at 30% ethanol up to 100% ethanol (2 min each).
  22. Soak in xylene 3 x 5 min.
  23. Mount with Permount (Fisher # SP15-100): put some above the section on the slide and press slowly onto the section with a coverslip without air bubbles. Do not move the coverslip until completely dry, which takes ~ 24 hours.

Materials

Name Company Catalog Number Comments
Xylene Fisher Scientific X3S-4 Change xylene every month depending on use. Xylene is TOXIC.
100% ethanol Fisher Scientific A406-20 Use to make EtOH gradient.
DPBS Dulbecco’s modified Phosphate Buffered Saline WITH Ca2+ and Mg2+
Na-Citrate buffer 10 mM, pH 6.5
H2O2 Sigma-Aldrich H-1009 30% stock => Dilute to 1% in DPBS
Blocking solution DPBS + 5% bovine serum albumin (BSA) + 0.1% Na Azide + 5% serum.
BSA
Sodium Azide NaAzide
avidin/biotin blocking kit Vector Laboratories SP-2001
Ab buffer DPBS + 2% BSA + 0.1% Na Azide + 2% serum (same as used for the blocking step)
ABC kit Vectastain PK-6100 Vector Laboratories
DAB peroxidase substrate Vector Laboratories SK-4100
ddH2O
Hematoxylin Fisher Scientific CS401-D counterstain
Permount Fisher Scientific SP15-100
Slides and cover-slips glass

DOWNLOAD MATERIALS LIST

Comments

28 Comments

  1. What is the purpose of dehydrating in ethanol and soaking in xylene before mounting the coverslips?

    Reply
    Posted by: Anonymous
    October 1, 2008 - 4:06 PM
  2. By dehydrating and soaking in xylene, the tissue is properly dehydrated prior to application of permamount. Without this step, the permamount would take longer to dry, and the coverslip would most likely slide during visualization. I've also noticed that visualization of the slides prior to curing of the permamount increases the amount of air bubbles formed when the slide is over the lamp.

    Reply
    Posted by: Anonymous
    October 20, 2008 - 5:29 PM
  3. purpose of citrate wash  

    Reply
    Posted by: Anonymous
    March 18, 2009 - 4:53 AM
  4. Boiling the sample in the sodium citrate solution allows for heat induced antigen retrival. However, this step is not always necessary, and is highly dependent upon the antigen or epitope you are attempting to stain for.

    Reply
    Posted by: Anonymous
    May 19, 2009 - 6:01 PM
  5. I have a problem with ABC kit, because it is expired, how many time can i use the kit after it expired. If i put the unmasking in hot bath and it is in boiling point, the results change? what is the chemistry principle of unmasking use.
    And i habe trouble with GFAP because it has nonespecific marking with neurons and don´t mark astrocites i think that is the time of incubation of primary antibody. but what can I do?

    Reply
    Posted by: Anonymous
    May 19, 2009 - 3:29 PM
  6. Hi Karolina, I am from Vector Labs, manufacturer of the ABC kit described in the video. My name is Craig Pow and I head the technical service department. We were invited to respond to your inquiries by the management at JoVE to perhaps offer you some insightful information as we are very familiar with the product of course. You have written essentially three inquiries. From our perspective, none of them are particularly in reference to the video and two of them do not pertain to the Vectastain ABC detection reagents. Regardless, we hope the following information is helpful. The Vectastain ABC kits are given a guaranteed workable shelf-life of 1² months from the time of receipt. We know the kits will work well without any loss of sensitivity or performance during this time period when stored appropriately at 4 degrees Celcius. It has been our experience, and that of many investigators, that this one year timeframe is by no means a "drop dead" date. In other words, the ABC kits can be used well beyond this one year date, in some cases two to three years, again without any loss of performance. The reagents in the kits are very stable under the refrigerated conditions and this is true for the numerous Vectastain ABC kits we offer that consist of concentrated and Ready-To-Use formats with different enzyme systems. You do not state which Vectastain ABC kit you have or how long the product has been expired, so I do not think we can provide you with further specifics. The following recently published references should provide you with details and current opinions regarding antigen unmasking in immunohistochemistry: 1) Shi, S.R. et al (²007) J. Histochem. Cytochem. 55(²):105-109 ²) Leong, T. Y-M. and Leong, A. S-Y. (²007) Adv. Anat. Pathol. 14(²):1²9-131 3) D'Amico, F. et al (²009) J. Immunol. Methods 341(1-²):1-18 Your third inquiry appears to be more of a question concerning specificity of the primary antibody rather than a background staining issue. Indeed primary antibodies directed against GFAP (glial fibrillary acidic protein) should bind to astrocytes. From your stated observations the primary antibody is not recognizing the correct cell type in your tissue. I am unclear if you are applying antigen unmasking techniques in this application. Certainly inappropriate antigen unmasking parameters (such as the salt used, pH, temperature, time) may alter tissue properties to generate inaccurate staining. This is where positive and negative controls would be vitally important to validate the staining results on the test sections. Primary antibody incubation times for standard sections cut at < 10 micrometers usually run around 30 to 60 min at room temperature. In some cases overnight incubation at 4 degrees Celcius is required where poor affinity or avidity for the target antigen occurs. We do offer a Troubleshooting Guide in pdf format on our website that describes controls to use in the absence of staining or if background staining is a problem. We hope you find this Guide and the information presented here helpful. Regards, Craig Pow      

    Reply
    Posted by: Anonymous
    May 21, 2009 - 4:20 PM
  7. I wish to know what is the concentration of haematoylin you prepared? Is it Myer's or Gill's?

    Reply
    Posted by: Anonymous
    May 21, 2010 - 12:04 PM
  8. The hematoxylin solution is Gill's #², diluted 1:1 with dd water. The catalog number from Fisher is: CS401-1D

    Reply
    Posted by: Anonymous
    August 4, 2010 - 1:42 PM
  9. I also wish to know what is the most proper antibody dilution to use initially to try

    Reply
    Posted by: Anonymous
    May 21, 2010 - 12:08 PM
  10. The concentration depends on the antibody you are using. Check the packing information. Appropriate concentrations can range from 1:100 to 1:100,000. I would initially try 1:100, then work with more dilute solutions.

    Reply
    Posted by: Anonymous
    August 4, 2010 - 1:30 PM
  11. Tissue in parrafin block has been kept in fridge for 8 months. Can I still use for immunohistochemistry staining of p53?

    Reply
    Posted by: Arifah A.
    May 27, 2010 - 4:36 AM
  12. Was the sample formalin treated prior to paraffin embedding? If so, the sample should be fairly stable, but the only way to confirm would be to use controls while staining your experimental sections.

    Reply
    Posted by: Anonymous
    August 4, 2010 - 1:35 PM
  13. My Vectastain elite ABC kit and DAB substrate had expired in August ²009. Can I still use them as I only need small quantity for my samples?
    Regards
    Yassir

    Reply
    Posted by: Anonymous
    August 4, 2010 - 12:06 AM
  14. I would contact Vector labs regarding the stability of their product post their printed expiration date.

    Reply
    Posted by: Anonymous
    August 4, 2010 - 1:37 PM
  15. The timing shown here in this video differs from the timing of protocols given in Vector Lab protocols.
    Please advise

    Reply
    Posted by: Anonymous
    October 7, 2010 - 9:31 PM
  16. I would follow the Vector labs timing, then adjust as needed if the results are not clear.

    Reply
    Posted by: Anonymous
    April 24, 2011 - 12:54 PM
  17. queria v~e em portugu&#²34;s n&#²²7;o entendo ingl&#²34;s

    Reply
    Posted by: Anonymous
    April 11, 2011 - 11:49 AM
  18. Olá Cely Granja de Oliveira,

    No momento nós não temos traduções disponíveis em Português. Sinto muito. Você pode utilizar os serviços de Tradução de Google para traduzir o texto.

    Obrigado,
    -Chris

    Reply
    Posted by: Chris M.
    April 11, 2011 - 12:30 PM
  19. give me information about acute lymphoblastic leukemia(ALL)?by RT(Reverse transcriptase PCR) or role of RT PCR in acute lymphoblastic leukemia(ALL)?

    Reply
    Posted by: Anonymous
    April 24, 2011 - 9:54 AM
  20. Can any one send me the drawing of the taste sensors in tong with, example: salt, sweet, sour, bitter & umami?

    Reply
    Posted by: Anonymous
    April 30, 2011 - 10:35 PM
  21. How much secondary biotynilated antibody should I use if I use 1:100 dilution of the primary? I cannot figure it out with the drops...

    Reply
    Posted by: Anonymous
    July 26, 2011 - 12:40 PM
  22. Many commercially available secondary antibodies have information indicating the effective concentration for different applications. For example, Western Blotting may require a 1:10,000 dilution, while an ELISA may require a 1:1000 dilution. You can test different concentrations on your samples to assure that you're not at the plateau for signal response. Use enough volume to cover the sample. Use of more volume will not change the secondary antibody concentration.

    Reply
    Posted by: Anonymous
    July 26, 2011 - 1:26 PM
  23. Although I have done IHC in a variety of forms in the past, this excellent video was ideal as a refresher. Victor--thank you so much for your clear, patient and very thorough description of the technique. I will be using a PAP pen for the first time soon and it was extremely helpful to watch you demonstrate how to use it.

    Reply
    Posted by: Anonymous
    August 16, 2011 - 11:52 AM
  24. Hi,
    I have questions: first is the antigen retrival, I'm using proteinase K from DAKO icubation time (3 min) instaed of heating process, however, it seems that the tissue morphology somehow changed my question is is that because the incubation time? or because of the concentration?

    my second question: In the dehydration step before mounting the coverslips, if the tissue happened to dry-out completely before mounting the coverslips would that affect the tissue or not?

    Thank you

    Reply
    Posted by: Anonymous
    October 4, 2011 - 5:00 PM
  25. Proteinase K usage for antigen retrieval is sensitive to time and concentration due to over digestion. Changes is tissue morphology could be attributed to either.
    Allowing the tissue to dry out completely prior to mounting may affect the morphology of the tissues dependent on the tissue being analyzed.

    Reply
    Posted by: Anonymous
    November 7, 2012 - 8:11 PM
  26. Is the coplin jar polypropylene or can glass??

    Reply
    Posted by: Marcia L.
    July 18, 2012 - 2:26 PM
  27. Not sure if our jars were polypropylene or polyethylene, but we have used them and glass coplin jars without any significant difference.

    Reply
    Posted by: Anonymous
    November 7, 2012 - 8:00 PM
  28. Hi, I have a trouble with my slides, il decollate after one hour incubation with antibodies:antibodies used are anti insulin and tissu is pancreas !! Can I have an explanation please especially when an H&E coloration has been successful with the same slides !!

    Reply
    Posted by: yesmine B.
    October 19, 2014 - 3:44 PM

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