Immunohistochemie: Paraffinecoupes Met behulp van de Vectastain ABC Kit van 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

Immunohistochemie (IHC) is een waardevolle techniek gebruikt om te lokaliseren / eiwit expressie te visualiseren in een gemonteerde weefselsectie met behulp van specifieke antilichamen. Er zijn twee methoden: de directe en indirecte methode. In dit experiment zullen we alleen maar beschrijven het gebruik van indirecte IHC kleuring. Indirecte IHC kleuring maakt gebruik van zeer specifieke primaire en biotine-geconjugeerd secundair antilichamen. Primaire antilichamen worden gebruikt om discreet eiwitten van belang te identificeren door te binden aan een specifiek epitoop, terwijl de secundaire antilichamen af ​​te trekken voor niet-specifieke achtergrond kleuring en versterken het signaal door het vormen van complexen aan de primaire antilichaam. Dia's kunnen worden gegenereerd op basis van vriescoupes of paraffine ingebedde coupes gemonteerd op glasplaatjes. In dit protocol, bespreken we de voorbereiding van de paraffine ingebedde coupes door het verwijderen, hydratatie met behulp van een gradiënt van alcohol, warmte geïnduceerde antigen retrieval, en het blokkeren van endogene peroxidase activiteit en niet-specifieke bindingsplaatsen. Sommige delen worden vervolgens gekleurd met antilichamen die specifiek zijn voor T-cel marker CD8 en terwijl anderen zijn gekleurd voor tyrosine hydroxylase. De dia's worden vervolgens behandeld met de juiste secundaire antilichamen geconjugeerd aan biotine, ontwikkelde vervolgens gebruik te maken van avidine-geconjugeerd mierikswortelperoxidase (HRP) met Diaminiobenzidine (DAB) als substraat. Na ontwikkeling worden de dia's tegengekleurd voor contrast, en gemonteerd onder dekglaasjes met Permount. Na voldoende droging, zijn deze dia's zijn dan klaar voor de beeldvorming.

Protocol

Kleuring van paraffine secties

  1. Dewax dia's met xyleen (Fisher X3 S -4) 3x gedurende 5 minuten elk (verandering xyleen elke maand afhankelijk van het gebruik, xyleen is giftig). Gebruik gerechten en covers voor 20 dia's van Fisher ref 08-812-1A en rekken gemaakt om deze gerechten te passen. 200 ml vloeistof per schotel.
  2. Hydrate door alcohol gradiënt als volgt uit (wijzigingen gradiënt om de 2 weken):
    • 2x in 100% ethanol (Fisher # A406-20) gedurende 2 minuten per
    • 2x in 95% ethanol gedurende 2 minuten per
    • 1x in 70% ethanol gedurende 2 minuten
    • 1x in 50% ethanol gedurende 2 minuten
    • 1x in 30% ethanol gedurende 2 minuten
    • 1x DDH 2 O gedurende 2 minuten
  3. Dompel onder in DPBS gedurende 5 minuten (DPBS = gewijzigd fosfaat Dulbecco's gebufferde zoutoplossing met Ca 2 + en Mg 2 +).

Antigen herstel:

  1. Verwarm de Na-citraat buffer (10 mM, pH 6,5) in de magnetron.
  2. Kook de dia's voor 15 min in de magnetron. De dia's mogen nooit droog dus check iedere minuut en voeg Na-Citraat wanneer dat nodig is.
  3. Laat afkoelen op kamertemperatuur.
  4. Block endogene peroxidase-activiteit met 1% H 2 O 2 in DPBS gedurende 20 min (1,5 ml van 30% aandelen Sigma H-1009 in 50 ml DPBS).
  5. Dompel onder in DPBS 3 x 5 minuten.
  6. Blokkeer niet-specifieke sites door incubatie overnacht bij +4 ° C in DPBS + 5% bovine serum albumine (BSA) + 0,1% Na Azide + 5% serum.

    Let op: Keuze van serum zal afhangen van de soorten waarbij antilichamen gebruikt voor het kleuren werden gemaakt. Blok met serum van de soorten waarbij de secundaire Ab (geconjugeerd aan biotine) werd gemaakt. Als dit niet beschikbaar is, gebruik serum van een andere soort dan van de een, waarin de primaire Ab werd gemaakt.

  7. Blokkeer de biotine sites met de avidine / biotine blokkeren kit (Vector laboratoria catalogus # SP-2001):
    • Incubeer met avidine D gedurende 15 minuten. Spoel kort met DPBS.
    • Incubeer met de biotine-oplossing gedurende 15 minuten.
  8. Incubeer in het primair Ab gedurende 2 uur bij kamertemperatuur, in DPBS + 2% BSA + 0,1% NaAzide + 2% serum (dezelfde als voor het blokkeren van stap).
  9. Soak in PBS 3 x 5 minuten.
  10. Incubeer met de secundaire Ab geconjugeerd met biotine gedurende 1 uur bij kamertemperatuur in DPBS + 2% BSA + 0,1% Na Azide + 2% serum (zelfde als gebruikt voor het blokkeren stap).
  11. Bereid ABC reagens op hetzelfde moment, omdat het te ontwikkelen voor ten minste 30 minuten voor gebruik! (Zie ook Materiaal)
  12. Bereid in een 50 ml conische buis. In 15 ml DPBS (geen serum, geen azide) voeg 3 druppels reagens A, mix en voeg 3 druppels reagens B en meng. Vermijd knijpen de flessen, in plaats van wachten tot de druppels te vormen op hun eigen. ABC kit Vectastain PK-6100 van Vector Labs.
  13. Dompel onder in DPBS 3 x 5 minuten.
  14. Incubeer met ABC reagens voor 30-45 min bij kamertemperatuur.
  15. Dompel onder in DPBS 3 x 5 minuten.
  16. Bereid DAB peroxidase substraat (Vector Labs # SK-4100) in 5 ml DDH 2 O in een glazen flacon onmiddellijk vóór gebruik (zie ook Materiaal)
    • 2 druppels buffervoorraad oplossing, mengen
    • 4 druppels van DAB, mix (zou moeten worden een beetje bruin)
    • 2 druppels H 2 O 2, mix
  17. Drop de DAB substraat op de top van de dia's en let op bruine vlekken.
  18. Dompel dia's in ijs + leidingwater om de reactie te stoppen.
  19. Afspoelen onder koud leidingwater gedurende 5 minuten.
  20. Tegenkleuring met hematoxyline (20 sec; Fisher CS401-D) en spoelen in leidingwater totdat er water uit te wissen.
  21. Uitdrogen door alcohol gradiënt te beginnen bij 30% ethanol tot 100% ethanol (2 min. elk).
  22. Soak in xyleen 3 x 5 minuten.
  23. Mount met Permount (Fisher # SP15-100): zet een aantal boven het gedeelte op de dia en druk op langzaam op de afdeling met een dekglaasje zonder luchtbellen. Beweeg niet het dekglaasje tot het volledig droog, dat duurt ~ 24 uur.

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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