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Post subject: CD4 (4B12) on iViewDAB
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Dear All,
Title: CD4 (4B12) on iViewDAB
My colleague has problem on IHC of CD4 clone 4B12 (Novocastra) by Ventana NexES IHC with iViewDAB-paraffin protocol (human FFPE). Staining on tonsil seems too weak. I would like to ask someone who have any experience about this Ab on Ventana iViewDAB protocol.
We know there is another successful Ab for CD4, clone 1F6 and we are wondering that we have to change to (try) clone 1F6.
I would greatly appreciate any advice.
Thanks,
sagins
Protocol
<Epitope retrieval>
Buffer: Tris-EDTA, pH9
Temperature: 125 deg/C, 3 min (PASCAL, Dako)
<Staining>
Antibody dilution: 1:100 with amplifier
Diluent: 1% BSA - 1/2TBS (pH 6.0)
Avidin - Biotin blocker is used
Incubation time: 32 min
We have tried severeal Ab diluents (Ventana Ab diluent, 1% BSA-TBS (pH 7.4), 1% BSA - 1/2TBS (pH 7.4), 1% - 1/2TBS (pH 6.0), Renoir red diluent (Biocare Medical), van Goch Yellow diluent (Biocare Medical) and 1% BSA - 1/2TBS (pH 6.0) looks best.
When Ab is concentrated than 1:100, only background staining increase. |
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 Tue Feb 03, 2009 10:13 am |
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Sorry i cant help reg Ventana.
I have just started to run IviewRed on a few ab.
We use both 1F6 and 4B12. Both works great using the same retrievals etc, i find TE ph 9 to work good on these. I use 4B12 at 1:600 and 1F6 at 1:200 dilution. I remember a 1F6 antibody that for no apparent reason suddenly stopped working properly - it was not caused by the end peroxidase blocking that might effect the epitope detected by 1F6.
have you compared with manual-staining or other detectionsystems?
Occationally (rare) we have found that ab have just stopped working properly - so we had to order a new lot.
Not much help really. I know there are some Ventana-users here. Since the site have been down it might take a few days until they drop by.
Ole |
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| Thu Feb 05, 2009 3:17 pm |
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Thanks so much for information, ole.
We haven't run manual IHC before, so I'll try manual IHC for testing Ab I have. Your dilution ratio is very helpful.
Ventana iViewDAB kit seems relatively (very) insensitive method even in cytoplasmic / membranous antigen, regarding results from other institutes (referring "IHC Protocols" in ths site and NordiQC website). Dilution ratio of CD4 clone 1F6 by iViewDAB may be extremely low (concentrated) and that's the reason we are trying 4B12.
sagins |
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| Thu Feb 05, 2009 11:39 pm |
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Post subject: CD4 (4B12) manual IHC
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Hmmm... No reply from Ventana users.
We might be doing silly way to stain CD4. We will consider changing Ab to 1F6.
However, I've done manual IHC of 4B12. Ab seems working but not my my manual IHC seems not strong enough, comparing to ole's (1:600).
4B12 dilution rate at 1:400 (MACH4 detection) produced somewhat staining, but seems not sufficient (please see picture; I'm always posting insufficient staining lol).
Lymphocytes in germinal center and interfollicular area are stained but the stain is not intense and ring-like membranous staining (described at NordiQC). Also, though macrophages are stained, stain is weak.
What can I for ? Wash? Block?
Or is staining OK?
<Picture>
A. Tonsil-low power
B. Tonsil-high power
<protocol>
described elsewhere
http://www.immunoportal.com/modules.php?name=Forums&file=viewtopic&t=1395
(same as MSH-6)
Last edited by sagins on Sun Apr 26, 2009 12:55 am; edited 1 time in total |
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| Fri Feb 20, 2009 10:13 am |
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Post subject:
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The staining is too weak. The retrieval looks OK, the protocol. However my first guess is that the retrieval settings are not optimal for this marker, even tho i use almost the same.
I have 3 year old photo of a intestine - altho IMO the staining is better today. This phjoto is of the 1F6 clone.
Ill try CD4 on ventana next week - however i have only iviewred.
Ole
Image of 1F6 in intestine |
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| Sat Feb 21, 2009 3:21 pm |
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Post subject: Beautiful stain (sigh)!
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Thank you and I really appreciate your help, ole!
And thanks for picture of beautiful CD4 stain.
Inappropriate HIER - yupp, maybe you're right, ole.
I've just only tried TE buffer pH 9 and 125 deg/C retrieval, so I'd better try others like...
1. TE pH 9 97 deg/C 40min
2. Tris pH 10 119 deg/C 1min and 97 deg/C 40min
3. Dako Target retrieval solution pH 9 (found it in fridge; who bought this!?) 119 deg/C 1min and 97 deg/C 40 min.
(all by PASCAL)
With tonsil sample, I stained a case of peripheral T cell lymphoma at the same time.
Though I've got membranous staining, there was also tissue damage.
I'll try 119 deg/C 1 min instead of 125 deg/C 3min and see what will happen...
sagins |
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| Mon Feb 23, 2009 10:28 am |
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I have tried twice, but i have had little luck, getting CD4 working good with my AP-detectionsystem on the ventana.
I guess Hogne could help? , as he is very experienced reg IHC and ventana. Perhaps you should try to send him a pm Sagins?
Ole |
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| Wed Mar 25, 2009 1:36 pm |
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Post subject: Thanks
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Thank you so much ole, for trying CD4 on Ventana and your kind advice.
We have seen nice CD4 staining with clone 1F6 by Ventana iViewDAB on Benchmark, so we are now thinking of buying 1F6.
Though NexES is pretty different from Benchmark, we'd like to try 1F6 on NexES.
And when 1F6 fail , We'd like to ask Hogne's help.
On the other hand, my manual staning had problem (MACH4) for a while.
So I couldn't proceed my assesment of my manual 4B12 staining, and now, I have to work on other than IHC.
It may take time to improve my manual staing but I'd like to keep trying.
Thanks again for your help and I hope I can feedback here.
sagins |
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| Tue Mar 31, 2009 8:05 am |
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Post subject: Trying CD4 clone 1F6
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We couldn't obtain good result from clone 4B12, and so tried 1F6.
Thanks to all, result seems acceptable.
(Dilution rate 1:10 ... expensive run!)
We're now waiting for clone SP35.
Hope this clone will stain at higher dilution rate...
sagins
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<staining condition>
CD4, clone 1F6, (Leica Microsystems)
Antigen retrieval: Tris-EDTA pH9, 121 deg/C 1 min
Dilution 1:10
Detection: iView DAB - nonparaffin protocol, with amplifier, NexES autostainer (peroxidase block omitted)
Antibody incubation: 32 min
<Images>
All are tonsil of human FFPE tissue sections.
Follicle
Germinal center
Blood vessels
(Showing pseudoperoxidase (:make sense?) reactivity)
Neutrophil
(Showing endogenous peroxidsase, few cells positive.) |
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| Sun Aug 09, 2009 2:05 am |
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Post subject:
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Confirm Anti-CD4 is now available.
Some of my customers have tried it with excellent results.
CC1 std, incubation for 16-32 min depending on your platform should produce some good results.
Ref#: Ventana: 790-4423 Roche: 05552737001
BR
Pal |
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| Mon Jan 18, 2010 9:50 am |
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