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Immunohistochemistry (IHC) / Precipitation on slides when dehydrating
« Last post by NSabine on September 05, 2018, 05:10:30 PM »

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.

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
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?


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?
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.   
Immunohistochemistry (IHC) / Ph 6:00
« Last post by Histolab on June 01, 2018, 09:09:32 AM »

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
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.
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
Immunohistochemistry (IHC) / IHC to Determine Phase of Cell Cycle??
« Last post by BPatrick on April 06, 2018, 11:19:12 AM »

I was wondering if anyone had some insight into a good antibody to determine which phase of the cell cycle that tumor cells are in.

It would be great to see how much heterogeneity there is within a group of cells and which phases of the cell cycle they are in so that we can determine if chemotherapeutics are having more effect on certain stages of the cycle.

Any info on this would be so much appreciated!

Immunohistochemistry (IHC) / Adivce on IHC Staining pattern needed!
« Last post by EJosephine on March 31, 2018, 05:24:34 AM »

I am in desperate need of advice on how to best to interpret/describe the pattern of staining I have achieved through IHC staining on mouse brain tissue. Unfortunately I have no further time to further optimise the protocol and have got to write up and discuss what I am seeing for an important final year project.

I have been staining mouse brain tissue using primary antibodies raised in mouse, trying to detect microglia using an anti-CD45 antibody (the best marker we had available) and a membrane-bound protein EpHA1 (the expression of which is not yet defined in the brain.)
To try and overcome the problems of my antibodies interacting with endogenous mouse Ig, I have used a M.O.M detection kit, which utilised an ABC detection method. I have used Bloxall to block endogenous enzyme activity, alongside avidin/biotin blocking.
I have obtained a similar pattern of staining for all of my primary antibodies, including my supposed negative control (reported to react with human and not mouse tissue.)
I have attached a link to some of the images from these experiments: (description of image is in the info for each picture)
Would this be best described as "non-specific, diffuse staining?" seeing as all antibodies are eliciting a similar picture? The "blank" slides incubated with the secondary antibody only show a cleaner picture, but still have some background staining.
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