Immunohistochimie: coupes en paraffine utilisant le kit de Vectastain ABC 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).

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Abstract

L'immunohistochimie (IHC) est une technique intéressante utilisée pour localiser / visualiser l'expression des protéines dans une section de tissu monté en utilisant des anticorps spécifiques. Il existe deux méthodes: la méthode directe et indirecte. Dans cette expérience, nous allons seulement décrire l'utilisation de l'IHC coloration indirecte. Indirects coloration IHC utilise très spécifiques primaires et de la biotine-anticorps secondaires conjugués. Les anticorps primaires sont utilisés pour identifier discrètement des protéines d'intérêt en se liant à un épitope précis, alors que les anticorps secondaires soustrait pour la coloration de fond non spécifique et d'amplifier le signal en formant des complexes à l'anticorps primaire. Les diapositives peuvent être soit générée à partir de coupes congelées, ou des sections paraffine montés sur lames de verre. Dans ce protocole, nous discutons de la préparation de paraffine sections par déparaffinage, l'hydratation en utilisant un gradient d'alcool, la récupération d'antigène de la chaleur induite, et le blocage de l'activité peroxydase endogène et non spécifiques des sites de liaison. Certaines coupes sont ensuite colorées avec des anticorps spécifiques pour le marqueur de cellules T CD8 et tandis que d'autres sont colorées pour la tyrosine hydroxylase. Les lames sont ensuite traitées avec des anticorps appropriés secondaire conjugué à la biotine, puis développé en utilisant de l'avidine-peroxydase conjugué de raifort (HRP) avec Diaminiobenzidine (DAB) comme substrat. Après développement, les diapositives sont contre pour le contraste, et montées sous lamelles avec Permount. Après un séchage adéquat, ces lames sont ensuite prêtes pour l'imagerie.

Protocol

Coloration des coupes à la paraffine

  1. Déparaffinage diapositives avec du xylène (Fisher X3 S -4) 3x pendant 5 minutes chacun (xylène changent chaque mois selon l'utilisation, le xylène est toxique). Utilisez de la vaisselle et les couvertures pour 20 diapositives de Fisher ref 08-812-1A et étagères faites pour s'adapter à ces plats. 200 ml de liquide par plat.
  2. Hydrater par gradient de l'alcool comme suit (changement de gradient toutes les 2 semaines):
    • 2x dans l'éthanol 100% (Fisher # A406-20) pendant 2 min de chaque
    • 2x dans l'éthanol 95% pendant 2 min de chaque
    • 1x dans l'éthanol 70% pendant 2 min
    • 1x dans l'éthanol 50% pendant 2 min
    • 1x dans l'éthanol 30% pendant 2 min
    • 1x ddH 2 O pendant 2 min
  3. Faire tremper dans du DPBS pendant 5 min (DPBS = phosphate de Dulbecco modifié saline tamponnée avec du Ca 2 + et Mg 2 +).

La récupération Antigène:

  1. Préchauffer le tampon Na-citrate (10 mM, pH 6,5) dans le micro-ondes.
  2. Cuire les diapositives pendant 15 min au micro-ondes. Les diapositives doivent jamais à sec afin de vérifier toutes les minutes et ajouter Na-citrate, si nécessaire.
  3. Laissez refroidir à température ambiante.
  4. Bloquer l'activité peroxydase endogène avec 1% de H 2 O 2 dans DPBS pendant 20 min (1,5 ml de bouillon de 30% Sigma H-1009 dans DPBS 50 ml).
  5. Faire tremper dans du DPBS 3 x 5 min.
  6. Bloc des sites non spécifiques par incubation une nuit à +4 ° C dans du DPBS + 5% de sérum albumine bovine (BSA) + 0,1% de Na azide + 5% de sérum.

    Remarque: Le choix de sérum dépendra de l'espèce dans laquelle des anticorps utilisés pour la coloration ont été faites. Bloc avec le sérum de l'espèce dans laquelle le secondaire Ab (conjugué à la biotine) a été faite. Si ce n'est pas disponible, l'utilisation de sérum provenant d'une espèce différente de celle dans laquelle l'Ac primaire a été rendu.

  7. Bloquer les sites de la biotine avec le kit avidine / biotine blocage (Vector Laboratories catalogue # SP-2001):
    • Incuber avec l'avidine D pendant 15 min. Rincer brièvement avec DPBS.
    • Incuber avec la solution de biotine pendant 15 min.
  8. Incuber dans l'enseignement primaire Ab pendant 2 heures à température ambiante, dans DPBS + 2% de BSA + 0,1% + NaAzide 2% de sérum (le même que pour le blocage étape).
  9. Faire tremper dans du PBS 3 x 5 min.
  10. Incuber avec l'Ab secondaire conjugué à la biotine pendant 1 heure à température ambiante dans DPBS + 2% de BSA + 0,1% Na azide + 2% de sérum (le même que celui utilisé pour l'étape de blocage).
  11. Préparer le réactif ABC dans le même temps, car elle doit se développer pendant au moins 30 min avant de les utiliser! (Voir Matériaux)
  12. Préparer dans un tube conique de 50 ml. En DPBS 15 ml (pas de sérum, aucun azoture) ajoutez 3 gouttes de réactif A, mélangez et ajoutez 3 gouttes de réactif B et mélanger. Ne pas tordre les bouteilles, au lieu d'attendre pour les gouttes pour former par leurs propres moyens. ABC kit PK-6100 Vectastain de Vector Labs.
  13. Faire tremper dans du DPBS 3 x 5 min.
  14. Incuber avec le réactif ABC pendant 30-45 min à température ambiante.
  15. Faire tremper dans du DPBS 3 x 5 min.
  16. Préparer substrat de la peroxydase DAB (Vector Labs # SK-4100) dans 5 ml ddH O 2 dans un flacon en verre juste avant son utilisation (voir Matériaux)
    • 2 gouttes de solution de stock tampon, mélanger
    • 4 gouttes de DAB, mélanger (devrait devenir légèrement brun)
    • 2 gouttes de H 2 O 2, mélanger
  17. Déposer le substrat DAB au sommet de la glisse et de regarder pour la coloration brune.
  18. Trempez glisse dans la glace + eau du robinet pour arrêter la réaction.
  19. Rincer sous l'eau froide du robinet pendant 5 min.
  20. Contre à l'hématoxyline (20 sec; Fisher CS401-D) et rincer à l'eau du robinet jusqu'à l'eau sort claire.
  21. Déshydrater dans l'alcool à partir de gradient éthanol à 30% jusqu'à 100% d'éthanol (2 minutes chacun).
  22. Faire tremper dans du xylène 3 x 5 min.
  23. Monter avec Permount (Fisher # SP15-100): mettre un peu au-dessus de la section sur la diapositive et appuyez lentement sur la section d'une lamelle, sans bulles d'air. Ne pas déplacer la lamelle jusqu'à séchage complet, qui prend environ 24 heures.

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

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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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