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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.
Neuroscience / Somatostatin IHC help
« Last post by SDashiell on March 05, 2018, 11:42:19 AM »
Has anyone successfully done an immunohistochemical stain against somatostatin? If so, could you kindly share your protocol, including specific primary and secondary antibodies?

Our lab has tried numerous protocol variations to no avail on 40 um mouse brain sections--we either get no signal or very patchy, light cell body staining near the outer edges of the sections. We have been using Millipore MAB354 primary and Invitrogen A11006 secondary.

If anyone could share some insight on staining this pesky protein, it would be very much appreciated!

Below is our latest IHC protocol:

3 x 20 min. wash in PBST
2 h blocking step
48 h Primary incubation at 4C (1:200 in PBST)
3 x 20 min. wash in PBST
4 h secondary incubation (1:500 in PBST)
3 x 20 min. wash in PBS

0.1% Triton X-100
in 1x PBS

Blocking solution:
0.1% Triton X-100
5% normal goat serum
0.2% bovine serum albumin
in 1x PBS

Thanks very much!

Immunohistochemistry (IHC) / Unspecific staining in skin
« Last post by DValerie on February 23, 2018, 01:45:07 PM »

I am doing IHC in paraffin-embedded tissues and I have problems with a unspecific staining. I work with equine wounds and I know that unspecific staining has been seen in sebaceous glands, but I have one also in the blood clot. I made a test to check if this was probably caused by the endogen avidin/biotin and I got a positive staining even without primary and secondary antibodies (I use ABC with alkaline phosphatase). So, do you guys think I should block endogen avidin/biotin or this may due to something else?

Thank you!

Immunohistochemistry (IHC) / Fc Fusion proteins used as IHC primary antibody
« Last post by CSuzanne on February 13, 2018, 11:32:49 AM »
Hi guys,

I am reviewing data from a CRO attempting to develop an IHC assay for us using a fusion protein and trying to detect its receptor via a standard IHC frozen tissue method with brief acetone fixation.  I don't believe it is working and am trying to find some literature on using Fc fusion proteins in this manner to better understand what conditions one needs to use, sensitivity of the assay, etc.   Does anyone have experience with this?

Thanks, Suzanne
Antibody C / Cyclin D1 Monoclonal Antibody (DCS-6)
« Last post by ZRossella on February 01, 2018, 09:58:53 AM »
Does anybody have any experience with Cyclin D1 Monoclonal Antibody (DCS-6) on Benchmark GX with ALK PHOS RED DETECTION KIT?
I've tried 1:50 and 1:100 diluition, CC from 0 to 30', with/without amplification and antibody diluent with/without casein block... slides going from a big cherry to a like-white tissue... making me crazy...

thanks in advance
I did an ICC and while imaging, I found that there are salt crystals on the coverslips. How can I avoid it?

My last steps are as follows:
After secondary, I wash the coverslips with PBS 3 times for 5 min.

I put a drop of my mounting media on a glass slide.

Take out the coverslip from the PBS, gently tap it on a kim wipe and place it on the glass slide.

Next, I keep the slides overnight at RT in dark and the following day I seal them using nail varnish.

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