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Immunohistochemistry (IHC) / Re: IHC artifact
« Last post by ABerisha on October 17, 2018, 11:20:30 AM »
hello RJason

Did you solve the problem with the AP artifact? because we have also the same issue with the Bond AP Kit.

Kind regards
2
Immunohistochemistry (IHC) / IHC artifact
« Last post by RJason on October 02, 2018, 09:48:22 AM »
Hello,

We are having artifact on our IHC slides. I am hoping someone on this forum has encountered this and can give us some advice.

We only see this on our red detection kit on our Leica bond. The brown is unaffected. The corresponding H&E slides are fine.
We have tried a number of things but we still can’t get consistent control of this issue.
Our rundown is standardized, we only use distilled water in our water baths, we take good care of our machine, clean the probe often, and refrigerate our red kits between runs.
Any ideas?

https://drive.google.com/open?id=1wUttTweP7SYLRM5pkGOBr0MBzCMrtJSA
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Immunohistochemistry (IHC) / Precipitation on slides when dehydrating
« Last post by NSabine on September 05, 2018, 05:10:30 PM »
Hello--

Hoping IHC experts can help with this dilemma:

When going from 70% ETOH --> 100% ETOH--> Citrosolv (citrus smelling xylene) there is progressive precipitation on the slide (milky waxy droplets/streaks esp when going fr 100%ETOH to Citrosolv). I've changed out all the ETOH and Xylene multiple times now.  Losing several slides and days of work each time this happens. 

Please help! Any input greatly appreciated.


Thanks!
4
Immunohistochemistry (IHC) / Tissue shredded after peroxidase IHC
« Last post by MAmanda on August 22, 2018, 03:50:37 PM »
Quick question. Why would brain slices be shredded after peroxidase IHC?

Two sets of tissue, both perfused (4%PFA) by different people. Both sets sectioned on cryostat by different people. Kept in antifreeze.
Both ran through peroxidase IHC in the exact same manner.
TBS (1x) washes, methanolic peroxide wash (15 min), TBS+ blocking wash. Overnight Primary antibody. Then washes, secondary, tertiary streptavadin incubation. DAB for blue-black reactivity.

One set of tissue was "shredded" at the end of the IHC. the other remained intact remarkably well. Trying to find out what could be compromising the structural integrity of the tissue either during perfusion or sectioning on the cryostat, as those are the only 2 times the tissue were treated differently. Thank you in advance for any comments
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Immunohistochemistry (IHC) / Black flakes on slide staining
« Last post by EAlberts on August 07, 2018, 10:00:41 AM »
Hi I am fairly new to IHC,

I have done some staining recently which has come out quite nicely, but recently I have had some that come out with black flakes on the top - does anyone know what this might be? Is it DAB precipitate or contamination from contaminated PBS?

Thanks,

Ellie
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Cell Markers Review / What cell types do not have IHC markers?
« Last post by DLuke18 on July 13, 2018, 02:32:56 PM »
My team is developing an assay to help with immune phenotyping and supplementing IHC.  Are there any specific cell types that do not have stains or are not detectible with IHC?
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General Histology / Butylacetate instead of xilen
« Last post by PAndres18 on July 10, 2018, 03:47:07 PM »
Hello everybody, I work in a laboratory for pathologists in Argentina, and we have an Thermo Scientific STP 120 automatic spin tissue processor. We are using xilene as the clearing agent, with two baths of xilene, 1 hour each. We are thinking of replacing xilene with butylacetate, but we donīt know how long we have to program each bath in order to have a good result. Does anybody work with this reagent? Thanks in advance for your cooperation.   
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Immunohistochemistry (IHC) / Ph 6:00
« Last post by Histolab on June 01, 2018, 09:09:32 AM »
Hi,

I will be perfusing rodent brains using 4% PFA and PBS at PH 6:00, but I am concerned that for IEg staing for C-fos and EGR-1 (Zif) the incubation medium is always used at PH 7.4, is the preparation of the tissue at PH 6.0 going to interfere with the c-fos/Zif ractivity?

Any advice will be appreciated
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Neuroscience / Re: Issue with cresyl violet staining of rat brain sections
« Last post by OBarbara on May 15, 2018, 01:47:07 PM »
Did you ever resolve the "curling" issue in your sections? I am experiencing the same curling with most of my sections, which are mouse brains, perfused with 4% para, cut on the vibratome at 70um, mounted on gelatin-subbed slides, air dried overnight and then run for Nissl. The protocol I use is the same as yours except the initial time in H20 is 10min, the time in Citrisolv is also 10min, and  the time for Nissyl stain is up to 1min. We then rinse twice in dH20, followed by 2min in 70% ETOH, differentiate for 4min, then 2min each of 95% ETOH and 100% ETOH, then twice for 5min in Citrisolv. I'm wondering if I need to shorten my times to mirror your times, but if you are still having curling issues, then I'm not sure that would help.
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General Discussion / degrease or not degrease?
« Last post by angela on April 12, 2018, 05:19:28 AM »
Hi, I have a general question concerning degreasing of sections. I've been asked to stain some frozen sections from human brain, fixed and cryo-protected. I was supposed to use the same protocol as some other colleagues did, and according to that, I should also use a degreasing step with 100% acetone for 45' before starting the staining. The reason for this step would be that we would like to see some antigens that would be located under the myelin. But I'm wondering if that is really necessary. I'm also concerned about some of the stainings that I would have to do, since some are receptors and I'm afraid they could be washed off by the degreasing step.
Any suggestion is welcome.
Thanks. Angela
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